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:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A heat-stable calmodulin binding protein was purified and characterized from the matrix of bovine heart mitochondria. It bound specifically to calmodulin in the presence of calcium, and strongly inhibited the stimulatory activity of calmodulin on phosphodiesterase. The estimated molecular weight of the calmodulin-binding protein was 61,000 dalton determined by SDS-polyacrylamide gel electrophoresis.
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PMID:Purification and characterization of the heat-stable calmodulin-binding protein from the matrix of bovine heart mitochondria. 687 Aug 76

Calmodulin was detected in dogfish erythrocyte lysates by means of phosphodiesterase activation. Anucleate dogfish erythrocyte cytoskeletons bound calmodulin. Binding of calmodulin was calcium-dependent, concentration-dependent, and saturable. Cytoskeletons consisted of a marginal band of microtubules containing primarily tubulin, and trans-marginal band material containing actin and spectrinlike proteins. Dogfish erythrocyte ghosts and cytoskeletons were found to contain a calcium-dependent calmodulin-binding protein, CBP, by two independent techniques: (a) 125I-calmodulin binding to cytoskeletal proteins separated by SDS PAGE, and (b) in situ azidocalmodulin binding in whole anucleate ghosts and cytoskeletons. CBP, with an apparent molecular weight of 245,000, co-migrated with the upper band of human and dogfish erythrocyte spectrin. CBP was present in anucleate ghosts devoid of marginal bands and absent from isolated marginal bands. CBP therefore appears to be localized in the trans-marginal band material and not in the marginal band. Similarities between CBP and high molecular weight calmodulin-binding proteins from mammalian species are discussed.
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PMID:The cytoskeletal system of nucleated erythrocytes. II. presence of a high molecular weight calmodulin-binding protein. 689 May 56

We examined a series of 2-aminochromone analogs typified by U-84569 [8-methyl-2-(4-morpholinyl)-7-(1-naphthylenylmethoxy)-4H-1- benzopyran-4-one] as potential antithrombotic agents. U-84569 proved to be a potent inhibitor of human platelet aggregation regardless of the agonist used. Subsequent experiments showed that U-84569 increased platelet cyclic AMP (cAMP) levels in intact cells, but U-84569 did not directly stimulate adenylate cyclase. Our experiments showed that U-84569 was a potent inhibitor of the low Km cAMP-dependent phosphodiesterase with an IC50 of 300 nM in platelet cytosol. Isobutylmethylxanthine had an IC50 of 10 microM in the same system. Although U-84569 elevated cAMP by inhibiting cAMP metabolism, we were interested in the mechanism by which cAMP blocked aggregation. Our first experiments showed that U-84569 concentration-dependently blocked agonist-stimulated, but not phorbol myristate acetate-dependent, phosphorylation of the 47 kDa protein kinase C substrate in platelets. These data suggested that U-84569 could interrupt receptor-mediated signal transduction. In support of this hypothesis, U-84569 proved to be a potent inhibitor of thrombin-stimulated inositol phosphate synthesis, diacylglycerol formation and Ca++ mobilization in intact cells. These data indicate that agonist-stimulated phospholipase C activity was reduced in U-84569-treated cells. There was no direct influence of U-84569 on either basal or thrombin-stimulated phospholipase C activity in broken cells, suggesting that U-84569 (by inhibiting phosphodiesterase and elevating cAMP), indirectly blocked receptor-mediated phospholipase C activation and aggregation in platelets. The 2-aminochromones represent a new class of potent antithrombotic agents.
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PMID:2-Aminochromones block human platelet aggregation by inhibiting cyclic AMP-dependent phosphodiesterase leading to reduced platelet phospholipase C activity. 768 15

The unique structures of process-bearing cells in the central nervous system (CNS) present an ideal model with which to study the differential distribution of mRNA. We conducted a side-by-side examination of the intracellular distribution of nine neural mRNAs by in situ hybridization histochemistry in mammalian brain and observed four general types of mRNA distributions. (1) Some mRNA species were confined to cell somas and included those encoding the glial proteins, myelin proteolipid protein and 2'3'-cyclic nucleotide-3'-phosphodiesterase and the neuronal enzymes, neuron-specific enolase and glutamate decarboxylase-67. (2) Some mRNAs were found abundantly within the cell soma and were also located throughout cellular processes. These included myelin basic protein (MBP) mRNA, which was localized to the cell soma and myelin sheaths of oligodendrocytes, and glial fibrillary acidic protein (GFAP) mRNA, which was localized to the cell soma and processes of reactive and some non-reactive astrocytes in the adult brain and radial glia in embryonic brain. (3) Some mRNAs were found primarily in perinuclear cytoplasm but in some cells were also observed in cell processes. These included mRNAs encoding the protein kinase C/calmodulin-binding substrates, RC3 (neurogranin) and GAP-43, which were identified in the somas as well as within the proximal dendritic branches of specific forebrain neurons. (4) Some mRNAs were localized primarily within cell processes. These included MAP2 mRNA, which was identified by deep staining within dendritic fields but by only light staining within neuronal cell bodies. The data also indicated that the stage of cellular development and the regional location of a cell within the CNS had a profound influence on translocation events. MAP2 mRNA was found in the dendritic processes of most neurons but was confined to the soma of neurons in specific brainstem nuclei. MBP mRNA was confined to the perinuclear cytoplasm of immature oligodendrocytes and was then transported into the myelin sheath at a developmental stage corresponding to myelination. The distribution patterns of these mRNAs are likely to reflect the mechanism by which the protein products of these molecules are targeted within neurons and glia. In addition, mRNA movement may be influenced by cellular and regional factors not encoded solely within the structure of the translocated mRNA.
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PMID:Cellular influences on RNA sorting in neurons and glia: an in situ hybridization histochemical study. 787 39

Parathyroid hormone (PTH)-mediated gene activation was assessed in the osteoblast-like rat cell line ROS17/2.8 with two PTH fragments harboring distinct activating domains: PTH-(1-34) and PTH-(28-48). The PTH response of genes expressed immediate early in the cell cycle or in the osteoblast developmental sequence was investigated. In addition, subtractive cloning was used to identify genes in ROS17/2.8 cells that are activated by the two PTH domains. PTH-(1-34) immediately increased the transcript levels of c-fos and c-jun at a considerably higher rate than PTH-(28-48). A significant immediate PTH effect on osteoblastic marker genes could not be detected, with the exception of elevated ornithine decarboxylase transcript levels. However, continuous application of PTH-(1-34) increased transcript levels of the osteoblast-specific osteocalcin gene and reduced those of other osteoblastic marker genes including alkaline phosphatase and the PTH/PTH-related peptide receptor. By subtractive cloning, nine cDNAs were isolated corresponding to mRNAs directly up-regulated by PTH-(1-34) or PTH-(28-48). Among these were a cyclic phosphodiesterase, a (cytosine 5)-methyltransferase, an 80-kDa protein kinase C substrate, junB, and a novel GC-binding protein. Three cDNAs are unknown at present. Interestingly, in all cases, the efficiency of gene activation by PTH-(28-48) was substantially lower in comparison with PTH-(1-34). PTH-mediated protein kinase C signaling in ROS17/2.8 cells may therefore constitute a minor pathway in comparison with the dominant cAMP/protein kinase A cascade.
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PMID:Domain-specific gene activation by parathyroid hormone in osteoblastic ROS17/2.8 cells. 870 88

A 19-amino acid residue peptide, Gly-Trp-Leu-Lys-Ile-Lys-Ala-Ala-Met-Arg-Trp-Gly-Phe-Phe-Val-Arg-Lys-Lys- Ala, corresponding to the basic amphiphilic alpha-helix (BAA) motif at the C-terminus of a recombinant tobacco calmodulin-binding protein, TCB60, was synthesized. The interaction of the synthetic binding domain with calmodulin (CaM) was analyzed by gel mobility shift assays, phosphodiesterase competition assays, and fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy. Mobility shift assays showed an apparent 2 kDa increase in CaM Mr in presence of synthetic peptide and CaCl2 in 4 M urea polyacrylamide gel electrophoresis. HPLC measurements of hydrolysis of cyclic AMP by CaM-dependent phosphodiesterase indicated the synthetic peptide competitively inhibits (Ki = 15-20 nM) stimulation of phosphodiesterase activity by CaM. Upon binding CaM, the fluorescence emission maximum of the synthetic peptide, which contained two tryptophanyl residues, shifted toward blue and increased in intensity. The circular dichroism spectra indicated the ellipticity of CaM increased at 208 and 222 nm upon complex formation with the synthetic peptide. 1H NMR studies showed that the peptide interacts with the aromatic residues in domains I and III of CaM. Taken together, these data provide direct evidence that the structurally conserved basic amphiphilic alpha-helix CaM-binding domain of the recombinant tobacco CaM-binding protein interacts with CaM at physiologically significant nanomolar concentrations and the microenvironments of both CaM and the synthetic binding domain are modified upon complex formation.
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PMID:Characterization of the basic amphiphilic alpha-helix calmodulin-binding domain of a 61.5 kDa tobacco calmodulin-binding protein. 904

Calmodulin-dependent cyclic nucleotide phosphodiesterase was identified in and purified to apparent homogeneity from the total calmodulin-binding protein fraction of bovine eye in a single step by immunoaffinity chromatography. The bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase is immunologically similar to the bovine brain 60-kDa isozyme. The purified enzyme had higher affinity for calmodulin than the 60-kDa phosphodiesterase isozyme from bovine brain, but similar affinity to that of the heart isozyme. When the Ca(2+)-dependence of the eye enzyme was compared to cardiac calmodulin-dependent cyclic nucleotide phosphodiesterase at an identical concentration of calmodulin, the bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase was activated at the same Ca2+ concentration as the bovine heart calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme.
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PMID:Calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine eye: high calmodulin affinity isozyme immunologically related to the brain 60-kDa isozyme. 905 31

The data presented in this report show that N-ethylmaleimide (NEM) is a powerful inhibitor of thrombin-induced platelet aggregation. NEM increased guanosine 3', 5'-cyclic monophosphate (cGMP) and adenosine 3', 5'-cyclic monophosphate (cAMP) levels in intact cells. The inhibition of cAMP high-affinity phosphodiesterase and cGMP phosphodiesterase was implicated in the elevation of the cyclic nucleotides. NEM dose dependently blocked the thrombin-stimulated, but not the phorbol myristate acetate-dependent phosphorylation of the protein kinase C substrate pleckstrin. Myosin light chain phosphorylation was also inhibited by NEM. In addition, the sulphydryl reagent inhibited Ca2+ mobilisation induced by thrombin. The data indicate that phospholipase C activation by thrombin is interrupted by NEM at the level of receptor-mediated signal transduction.
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PMID:N-ethylmaleimide inhibition of thrombin-induced platelet aggregation. 1048 31

Calmodulin-binding proteins are involved in numerous cellular signaling pathways. The biotinylated-calmodulin overlay is a nonradioactive method widely used to detect calmodulin-binding proteins in tissue and cell samples. This method has several limitations; therefore, we developed a nonradioactive calmodulin-binding protein detection overlay using an S-tag-labeled calmodulin fusion protein. An expression system was used to generate a calmodulin fusion protein with an S-tag label, a 15 amino acid sequence that binds to a 105 amino acid S-protein. The S-protein is conjugated to horseradish peroxidase for final detection with a chemiluminescent substrate. The S-tag calmodulin was compared to purified calmodulin and biotinylated calmodulin in a calmodulin-dependent phosphodiesterase assay. The results of the calmodulin-dependent phosphodiesterase assay indicate that S-tag calmodulin induces higher phosphodiesterase activity than biotinylated calmodulin and lower activity than purified calmodulin. A comparison of the biotinylated and S-tag calmodulin overlay assays indicate that S-tag calmodulin is more sensitive than biotinylated calmodulin in the detection of calcineurin, a known calmodulin-binding protein. The overlay assay results also indicate that the S-tag calmodulin and biotinylated calmodulin detect similar calmodulin-binding proteins in colon epithelial cells. In conclusion, the S-tag calmodulin overlay assay is a consistent, sensitive, and rapid nonradioactive method to detect calmodulin-binding proteins.
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PMID:Calmodulin-binding protein detection using a non-radiolabeled calmodulin fusion protein. 1135 39

It is now widely accepted that actions of intracellular Ca(2+) are mediated by a four-domain Ca(2+)-binding protein, calmodulin. Brain is especially rich in calmodulin, containing about 400 mg (24 ?mol) of EGTA-extractable calmodulin per kg of brain. However, only a fraction of the above amount is required for the calmodulin-activated enzymes and most of the rest may be assigned to calmodulin-binding proteins, proteins which are apparently devoid of enzyme activities but undergo Ca(2+)-dependent associations with calmodulin. Several of such proteins have been recently discovered in brain. These include a heat-labile 80 K phosphodiesterase inhibitor protein (calcineurin), a heat-stable 70 K phosphodiesterase inhibitor protein, a 50 K protein, myelin basic protein, tubulin, microtubule ? (tau) factor, a spectrin-like doublet protein (240 plus 235 K) (calspectin; fodrin) and a particle-associated 155 K protein. Functions of these calmodulin-binding proteins have not been fully elucidated yet. Some proteins may be calmodulin-regulated enzymes catalyzing yet unknown biochemical reactions, e.g. a protein phosphatase activity was found for calcineurin. Some proteins may interact with contractile elements or cytoskeleton of the cell, e.g. ? factor and calspectin interacted with tubulin and F-actin, respectively and tubulin itself is a calmodulin-binding protein. So, interesting possibilities are the regulation of the functions of cytoskeleton by calmodulin through these calmodulin-binding proteins. Regulation of microtubule assembly by Ca(2+)-dependent binding of calmodulin to tubulin and/or ? factor and possible involvement of calspectin in the mechanism regulating axonal transport of neuronal proteins have been suggested. Thus, the exploration of the regulating functions of Ca(2+)/calmodulin in brain depends largely upon the further study of the properties of these calmodulin-binding proteins.
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PMID:Calmodulin-binding proteins in brain. 2048 36


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