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)

We investigated the biochemical and functional properties of five vasopressin V2 receptor mutants (L44F, L44P, W164S, S167L, and S167T) that were recently described in families with a history of X-linked nephrogenic diabetes insipidus. COS.M6 cells transfected with cDNA encoding these mutants acquired < 4% specific [3H]arginine vasopressin (AVP) binding sites on the cell surface in comparison with cells transfected with cDNA coding for the wild-type receptor. Membrane preparations from COS.M6 cells or human embryonic kidney 293 cells expressing these mutants did not respond with an increase in adenylyl cyclase activity in response to AVP, which is in contrast to membranes from cells expressing the wild-type. By analyzing fusion proteins of the V2 receptor and Escherichia coli alkaline phosphatase attached to the carboxyl terminus of the receptor moiety, we found that the mutants L44P, W164S, S167L, and S167T lacked complex glycosylation and were expressed at low levels. The data suggest that the mutants L44P, W164S, S167T, and S167L are misfolded and therefore retained within the endoplasmic reticulum and degraded. In contrast, the fusion proteins carrying the mutant L44F and the in vitro mutant S167A were expressed in their mature form at wild-type levels; however, only the mutant S167A was functionally active. Site-directed mutagenesis of S167 revealed that elimination of the endogenous hydroxyl group (S167A) yielded a protein with properties identical to those of the wild-type receptor, whereas both the introduction of a methyl group (S167T) and the replacement of the hydroxyl group by an isopropyl group (S167L) profoundly disturbed receptor processing. The data show that minute changes at codon 167 nearly abolish expression of a mature protein, thus defining structural requirements of this codon.
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PMID:Vasopressin V2 receptor mutants that cause X-linked nephrogenic diabetes insipidus: analysis of expression, processing, and function. 886 26

Double stranded RNA-dependent protein kinase (PKR) is a double stranded RNA-activated, interferon-induced serine-threonine kinase that participates in both the antiviral and antiproliferative properties of interferon. We previously found that influenza virus inhibited PKR function by recruiting or activating a cellular inhibitor termed P58(IPK). The present study was undertaken to complement our earlier analyses, which demonstrated that P58(IPK) efficiently inhibited PKR autophosphorylation and activity in vitro. We now report that P58(IPK) down-regulates PKR and, in turn, stimulates protein synthetic rates inside the cell. Using transfection analysis, we show that P58(IPK) stimulates translation of secreted embryonic alkaline phosphatase reporter gene mRNA. Furthermore, we found that at least two regions of the P58(IPK) molecule were required for PKR inhibitory activity in COS-1 cells: (i) the DnaJ similarity region at the carboxyl terminus (amino acids 391-504); and (ii) the tetratricopeptide repeat 6 (TPR6) domain (amino acids 222-255) located in the middle of the P58(IPK) protein and within the eukaryotic protein synthesis initiation factor 2alpha homology region. P58(IPK) variants lacking either one of these regions were unable to stimulate secreted embryonic alkaline phosphatase protein synthetic rates. Consistent with this data is the observation that the DeltaTPR6 mutant (the P58(IPK) variant lacking the TPR6 motif) failed to block PKR activity in vitro. Based on these data and our earlier in vitro functional and PKR-P58(IPK) binding analyses, a revised model of PKR regulation by P58(IPK) is presented.
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PMID:The 58-kDa cellular inhibitor of the double stranded RNA-dependent protein kinase requires the tetratricopeptide repeat 6 and DnaJ motifs to stimulate protein synthesis in vivo. 891 May

N52 is a widely used monoclonal antibody reported to recognise both phosphorylated and non-phosphorylated forms of neurofilament (NF)-H. N52 is therefore classified as a phosphorylation-independent-type antibody. N52 is strongly reactive with NF-H in COS cells transfected with NF-H alone but co-transfection of NF-H with the neurofilament kinase cdk-5 and one of its activators p35, induced phosphorylation of NF-H that abolished this reactivity. Treatment of the cdk-5 phosphorylated NF-H with alkaline phosphatase so as to remove phosphate restored N52 reactivity. A fragment of NF-H containing the consensus cdk-5 sites was reactive with N52 but following co-transfection with cdk-5/p35 a slower migrating fragment species generated by cdk-5 was not labelled by N52. These results demonstrate that N52 is not a truly phosphorylation-independent-type NF-H antibody and suggest that the N52 epitope contains sites targeted for phosphorylation by cdk-5.
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PMID:Cellular phosphorylation of neurofilament heavy-chain by cyclin-dependent kinase-5 masks the epitope for monoclonal antibody N52. 891 96

Protein kinase D (PKD) is a serine/threonine protein kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour-promoting phorbol esters, membrane-permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat-1 cells), in secondary cultures of mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co-transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS-7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC-dependent signal transduction pathway.
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PMID:Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway. 894 45

Nidulal (1), a novel inducer of differentiation of human HL-60 promyelocytic leukemia cells, was isolated from fermentations of the basidiomycete Nidula candida together with low amounts of niduloic acid (2). Both compounds are bisabolane sesquiterpenes. Their structures were elucidated by spectroscopic methods. In reporter gene assays nidulal (1) preferentially activated the transcription factor complex AP-1-mediated expression of secreted alkaline phosphatase in COS-7 cells. In addition nidulal (1) and niduloic acid (2) exhibited weak cytotoxic and antibiotic activities.
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PMID:Nidulal, a novel inducer of differentiation of human promyelocytic leukemia cells from Nidula candida. 903 63

Leucocyte alkaline phosphatase (LAP) is an enzyme expressed on the external aspect of the neutrophilic granulocyte plasma membrane, and represents a specific marker for the fully differentiated granulocyte. In this report we characterize 1B12.1, a monoclonal antibody raised against human bone alkaline phosphatase, by its ability to recognize the LAP protein. As assessed by Western blot analysis, following electrophoresis under non-reducing conditions, the antibody specifically reacts with LAP upon forced expression of the protein in simian COS-7 fibroblasts. In addition, the 1B12.1 antibody recognizes partially purified LAP isolated from peripheral blood granulocytes. With this antibody we developed a quantitative flow-cytometry-based method for the determination of LAP. Double fluorescence flow cytometry demonstrated that the LAP protein was present in relatively high amounts in neutrophilic granulocytes, but not in monocytes, natural killer cells, or B and T lymphocytes of normal individuals. The protein was completely absent in granulocytes obtained from chronic myeloid leukaemia and paroxysmal nocturnal haemoglobinuria patients. Higher than normal levels of LAP protein were evident in neutrophilic granulocytes of patients suffering from polycythaemia vera, essential thrombocythaemia and severe aplastic anaemia. However, the highest amounts of LAP protein were present in the granulocytes of normal individuals treated with G-CSF for the isolation of peripheral blood stem cells.
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PMID:Flow cytometry of leucocyte alkaline phosphatase in normal and pathologic leucocytes. 907 26

Bone morphogenetic proteins (BMPs) are multifunctional proteins that comprise the largest subfamily of the transforming growth factor-beta. These proteins bind to types I and II serine/threonine kinase receptors. Ligand-induced heteromeric dimerization of these receptors is the key event in initiation of biological responses. We report here large-scale expression and purification of extracellular domain of the type I receptor for BMP-2/4, using a silkworm expression system. This soluble form of BMP receptor (sBMPR) was in monomer form in solution and bound to BMP-4 but not to activin or transforming growth factor-beta1. Surface plasmon resonance studies showed that kinetic parameters of sBMPR for BMP-4 consisted of a relatively rapid association rate constant (ka = 3.81 +/- 0.19 x 10(4) s-1 M-1) and an extremely slow dissociation rate constant (kd = 3.69 +/- 0.26 x 10(-4) s-1). From these two kinetic parameters, affinity was determined to be similar to that of the intact membrane-associated receptor expressed on COS cells. sBMPR inhibited the alkaline phosphatase activity in BMP responsive cell lines such as mouse osteoblastic cell MC3T3-E1 and bone marrow stromal cell ST2. These data indicate that the extracellular domain of type I receptor for BMP-2 and BMP-4 is sufficient for high-affinity binding to its ligands and should prove useful in understanding the role of BMP-2/4 in vivo, because a suitable high-affinity anti-BMP antibody has yet to be developed.
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PMID:Interaction between soluble type I receptor for bone morphogenetic protein and bone morphogenetic protein-4. 911 Oct 68

Resistance to the neomycin analogue G418 forms the basis of a dominant marker selection system for mammalian cells transfected with the bacterial neomycin gene. We found that COS-1 cells stably transfected with the neomycin resistance gene had a greater than 50% reduction in cell-associated glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP). A similarly reduced amount of AP was also observed in wild-type COS-1 cells incubated in the presence of G418 or other aminoglycoside antibiotics. The AP was released from cells into the culture supernatant in its GPI-anchored form. Our data suggest that the G418-induced reduction of AP involves a vesiculation process of COS-1 cells.
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PMID:Addition of G418 and other aminoglycoside antibiotics to mammalian cells results in the release of GPI-anchored proteins. 922 84

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is a secretory protein present in high amounts in mammalian body fluids. Its cDNA has been isolated and encodes a signal peptide of 23 amino acids and the mature protein of 816 amino acids. We generated cDNAs encoding a signal peptide-deficient and a GPI-anchored form of GPI-PLD and transiently transfected these constructs into COS-1 cells. The signal peptide-deficient form of GPI-PLD was expressed as a 90-kDa protein that was catalytically active and was localized intracellularly. Cells transfected with cDNA encoding the GPI-anchored form of GPI-PLD expressed a catalytically active enzyme of 100 kDa that could be labelled with [3H]ethanolamine demonstrating its modification by a GPI structure. Expression of the GPI-anchored form of GPI-PLD resulted in the release of endogenous GPI-anchored alkaline phosphatase from COS-1 cells, whereas expression of the intracellular form of GPI-PLD had no effect on membrane attachment of endogenous alkaline phosphatase. Similarly, in cells cotransfected with GPI-anchored placental alkaline phosphatase (PLAP) and the GPI-anchored form of GPI-PLD, PLAP was released into the cell culture supernatant while expression of the signal peptide-deficient form of GPI-PLD did not affect the amount of cell-associated PLAP.
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PMID:Expression of intracellular and GPI-anchored forms of GPI-specific phospholipase D in COS-1 cells. 926 57

In Escherichia coli, the topology of inner membrane proteins can be studied conveniently with the alkaline phosphatase/beta-galactosidase (PhoA/LacZ) gene fusion system. PhoA is enzymatically active only when fused to external domains, LacZ when fused to cytoplasmic domains. In eukaryotic cells, only time consuming methods exist to study the topology of membrane proteins. We have extended in the first systematic study the PhoA/LacZ gene fusion system originally developed for E.coli for use in eukaryotic COS.M6 cells. We have fused PhoA and LacZ to the putative external and cytoplasmic loops of rat aquaporin 2 (AQP2), for which a model with six transmembrane domains was proposed previously. The fusion proteins were expressed in E.coli and COS.M6 cells and immunoblot analyses and enzyme activity assays were performed to localize the protein domains in both cell types. The data obtained in E.coli correlated mostly with the predictions of the six transmembrane domain model. However, two fusions were found to exhibit both high PhoA and high LacZ activity, thereby complicating the construction of a complete AQP2 model. In COS.M6 cells, the PhoA fusions were inactive. In contrast, the LacZ fusions succeeded and showed an activity pattern in complete agreement with the predictions of the six transmembrane domain model. Therefore, LacZ fusions can localize cytoplasmic loops in COS.M6 cells by means of a simple enzymatic assay with high reliability and may be used in future studies to develop topological models of other eukaryotic membrane proteins in their authentic cell systems.
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PMID:Topology of eukaryotic multispanning transmembrane proteins: use of LacZ fusions for the localization of cytoplasmic domains in COS.M6 cells. 927 85


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