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)

1. Effects of atrial natriuretic peptide (ANP) on the L-type Ca2+ channels were examined in rabbit isolated ventricular cells by use of whole-cell and cell-attached configurations of the patch clamp methods. ANP produced a concentration-dependent decrease (10-100 nM) in amplitude of a basal Ca2+ channel current. 2. The inactive ANP (methionine-oxidized ANP, 30 nM) failed to decrease the current. 3. 8-Bromo-cyclic GMP (300 microM), a potent activator of cyclic GMP-dependent protein kinase (PKG), produced the same effects on the basal Ca2+ channel current as those produced by ANP. The cyclic GMP-induced inhibition of the Ca2+ channel current was still evoked in the presence of 1-isobutyl-3-methyl-xanthine, an inhibitor of phosphodiesterase. ANP failed to produce inhibition of the Ca2+ channel current in the presence of 8-bromo-cyclic GMP. 4. In the single channel recording, ANP and 8-bromo-cyclic GMP also inhibited the activities of the L-type Ca2+ channels. Both agents decreased the open probability (NPo) without affecting the unit amplitude. 5. The present results suggest that ANP inhibits the cardiac L-type Ca2+ channel activity through the intracellular production of cyclic GMP and then activation of PKG.
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PMID:Cyclic GMP-mediated inhibition of L-type Ca2+ channel activity by human natriuretic peptide in rabbit heart cells. 754 93

The synthesis of the neuropeptide precursor proenkephalin was measured in bovine adrenal chromaffin cells following radiolabeling with [35S]methionine. Treatment of chromaffin cells with pertussis toxin (100 ng/ml) approximately doubled proenkephalin synthesis without altering total protein synthesis. Pertussis toxin pretreatment also increased proenkephalin synthesis in chromaffin cells exposed to vasoactive intestinal peptide (VIP) and 3-isobutyl-1-methylxanthine (IBMX). Combinations of IBMX plus nicotine, VIP, or histamine also synergistically enhanced proenkephalin synthesis, with no further elevation when the cells were also pretreated with pertussis toxin. The action of forskolin, a direct activator of adenylate cyclase, on proenkephalin synthesis was similarly potentiated by pertussis toxin or IBMX, presumably reflecting the abilities of both the toxin and this phosphodiesterase inhibitor to enhance the cyclic AMP response to forskolin. In contrast, increased synthesis of proenkephalin in response to phorbol esters was not affected by pertussis toxin treatment. These results suggest that pertussis toxin potentiates proenkephalin synthesis primarily through inactivation of guanine nucleotide-binding proteins that inhibit adenylate cyclase, although other signaling pathways may also be involved.
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PMID:Pertussis toxin enhances proenkephalin synthesis in bovine chromaffin cells. 769 72

A novel plasmid was generated which allowed the expression of the cytosolic bacterial enzyme chloramphenicol acetyl transferase (CAT) in COS-7 cells. Upon transfection, the majority of the novel CAT activity was found in the cytosol fraction of COS cells. Chimeric molecules were made between N-terminal portions of the type IVA cyclic AMP-specific rat 'dunce-like' phosphodiesterase (RD1) (RNPDE4A1A; rPDE-IVA1) fused to CAT at its N-terminus. Expression in COS-7 cells of chimeras formed from 1-100RD1-CAT and 1-25RD1-CAT now showed CAT activity associated with the membrane fraction. In contrast, a chimera formed from 26-100RD1-CAT showed an identical expression pattern to native CAT, with the major fraction of CAT activity occurring in the cytosol fraction. Membrane-bound CAT activity provided by 1-100RD1-CAT and 1-25RD1-CAT was not released by either high-salt or washing treatments but was solubilized in a dose-dependent fashion by the non-ionic detergent Triton X-100. Subcellular fractionation of COS-7 cells showed that, as with RD1, the membrane-bound activity of the RD1-CAT chimera followed that of the plasma membrane marker 5'-nucleotidase. Plasmids containing chimeric cDNAs were exposed to a coupled transcription-translation system that, in addition to the full-length chimeras, was found to generate a range of N-terminal truncated species due to initiation at different methionine residues. Incubation of the mature protein products formed in this system with a COS cell membrane fraction showed that only those chimeric CAT constructs containing the first 25 amino acids of RD1 became membrane-associated. The unique 25 amino acid N-terminal domain of RD1 contains structural information that can confer membrane association upon an essentially soluble protein.
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PMID:Chimeric constructs show that the unique N-terminal domain of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1A; rPDE-IVA1) can confer membrane association upon the normally cytosolic protein chloramphenicol acetyltransferase. 777 57

A bone and cartilage enzyme with both 5'-nucleotide phosphodiesterase I and nucleotide pyrophosphohydrolase (NTPPPH) activity modulates physiologic mineralization and pathologic chondrocalcinosis by generating inorganic pyrophosphate. We hypothesized that, as for alkaline phosphatase, expression of an NTPPPH gene can be shared by cells from bone, cartilage, and liver and by certain leukocytes. Recently, we demonstrated the hepatocyte and murine plasma cell membrane glycoprotein PC-1 to have both 5'-nucleotide phosphodiesterase I and NTPPPH activity. We detected polypeptides cross-reactive with PC-1 in human U20S osteosarcoma cells, articular chondrocytes, homogenized human knee cartilages, human knee synovial fluids, hepatoma cells, and murine plasmacytoma cells. Constitutive low abundance PC-1 mRNA expression was detected in U20S cells and chondrocytes by a nested RNA-PCR assay and by Northern blotting. TGF beta is known to substantially increase NTPPPH activity in primary osteoblast cultures. We demonstrated that TGF beta 1 increased NTPPPH activity and the level of PC-1 mRNA and immunoprecipitable [35S]-methionine-labeled PC-1 polypeptides in U20S cells. The identification of PC-1 as an NTPPPH expressed in cells derived from bone and cartilage may prove useful in furthering the understanding of the role of NTPPPH i n physiologic and pathologic mineralization.
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PMID:Expression of the murine plasma cell nucleotide pyrophosphohydrolase PC-1 is shared by human liver, bone, and cartilage cells. Regulation of PC-1 expression in osteosarcoma cells by transforming growth factor-beta. 804 Mar 11

Calmodulin (CaM) has two hydrophobic surface patches that are particularly rich in Met residues, and these are the major contact areas where CaM interacts with its target enzymes. The amino acid Leu has been introduced by site-directed mutagenesis to replace all the Met residues in CaM. All nine individual Met-->Leu mutants of CaM as well as some double and quadruple mutants were expressed in Escherichia coli. All mutants could be purified by calcium-dependent hydrophobic affinity chromatography, indicating that they still expose their hydrophobic surfaces upon binding calcium. Each single Met-->Leu mutation in the C-terminal domain of the protein had little effect on its ability to activate phosphodiesterase (PDE), while a quadruple mutant with four C-terminal Leu residues instead of Met has a significantly lower affinity for PDE. The M36L mutant is a poor activator compared with the other three N-terminal single Met-->Leu mutants, which have a slightly lower affinity for PDE than wild-type CaM. The introduction of a positively charged Arg for Met-145 resulted in an almost complete loss of CaM's ability to activate PDE. Nuclear magnetic resonance spectroscopy was used to show that most CaM mutants retain their overall three-dimensional structure. Thus, the altered activation properties appear to arise from differences in the flexibility and polarizability of the Met and Leu sidechains, rather than from structural perturbations.
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PMID:The effect of Met-->Leu mutations on calmodulin's ability to activate cyclic nucleotide phosphodiesterase. 819 99

The effects of selective phosphodiesterase inhibitors, cyclic AMP (cAMP) elevating agents and stable analogues of cyclic nucleotides, on the release of arachidonate induced by N-formyl-Met-Leu-Phe (fMLP) were investigated on human peripheral blood mononuclear cells. The selective phosphodiesterase IV inhibitors, rolipram and Ro 20-1724, and the non-selective phosphodiesterase inhibitor, theophylline, elicited a concentration-dependent inhibition of arachidonate release (EC50 = 1.3 x 10(-6) M, 3.2 x 10(-6) M and 3.7 x 10(-4) M respectively). The selective phosphodiesterase III inhibitor, milrinone (10(-5) M), only caused a slight effect while the phosphodiesterase V inhibitor, zaprinast (10(-5) M), the beta 2-adrenoceptor agonists, salbutamol and fenoterol (10(-5) M), failed to inhibit arachidonate release. Forskolin (10(-5) M) and N6,2'-O- dibutyryladenosine 3':5'-cyclic monophosphate (db-cAMP), 10(-3) M) elicited a moderate inhibition. Forskolin increased the effects of rolipram and Ro 20-1724 (EC50 = 4.5 10(-7) M and 4 x 10(-7) M respectively). Incubation of the cells with rolipram (10(-8) to 10(-5) M), Ro 20-1724 (10(-8) to 10(-5) M, forskolin (10(-5) M) or salbutamol (10(-5) M) alone, induced a moderate increase or no increase at all in intracellular cAMP. However, in the presence of forskolin, rolipram (10(-8) to 10(-6) M) and Ro 20-1724 (10(-8) to 10(-6) M) induced significant and concentration-dependent increase in intracellular levels of cAMP. These results suggest that the potent inhibition of arachidonate release from mononuclear cells by selective phosphodiesterase IV inhibitors may be due to increases in discrete pools of intracellular cAMP.
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PMID:Involvement of cyclic AMP in the effects of phosphodiesterase IV inhibitors on arachidonate release from mononuclear cells. 856 80

In photoreceptor cells, visual transduction occurs through photoexcitation of rhodopsin, GTP activation of the alpha subunit of transducin, and interaction between GTP-bound transducin alpha subunit and the inhibitory gamma subunit of phosphodiesterase. The gamma subunit of phosphodiesterase, in turn, accelerates the hydrolysis of GTP on the alpha subunit of transducin. Within the COOH-terminal residues (46-87) of the phosphodiesterase gamma subunit, Trp-70 has been implicated in phosphodiesterase activation, transducin alpha subunit-phosphodiesterase gamma subunit interaction, and the GTP hydrolysis accelerating activity. We have derivatized the phosphodiesterase gamma subunit with a reversible photoactivatable reagent, [125I]N-[(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) ]cysteine ([125I]ACTP), at cysteine (Cys-68). A light-dependent, cross-linked complex of guanosine 5'-(gamma-thio)triphosphate-bound transducin alpha subunit and ACTPderivatized phosphodiesterase gamma subunit formed after photolysis of a 1:1 stoichiometic complex of the two proteins. The specificity of complex formation between the transducin alpha subunit and the phosphodiesterase gamma subunit was demonstrated by specific protection by the C68A mutant of the phosphodiesterase gamma subunit. The cross-linked complex was treated with beta-mercaptoethanol to transfer the 125I photomoiety from the phosphodiesterase gamma subunit to the transducin alpha subunit. Combined techniques involving electrophoresis, chemical and enzymatic cleavage, and chemical and radiosequencing were used to identify photoinsertion sites on the alpha3 and alpha4/beta6 regions of the transducin alpha subunit. Three photo-labeled residues, His-244 (alpha3 helix), Met-308, and Arg-310 (alpha4/beta6 interface), were specifically identified as photoinsertion sites. Utilizing the crystal structure coordinates of the GTP-bound transducin alpha subunit and molecular modeling, we conclude that Cys-68 of the phosphodiesterase gamma subunit is located at a position between the exposed face of the alpha3 and alpha4 helices of the transducin alpha subunit. We propose that the phosphodiesterase gamma subunit interacts with GTP-bound transducin alpha subunit at multiple sites in which the cysteine 68 to tryptophan 70 sequence of the phosphodiesterase gamma subunit, which is critical for GTP hydrolysis accelerating activity, interacts in the alpha3/alpha4/beta6 region of GTP-bound transducin alpha subunit.
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PMID:Interaction sites of the COOH-terminal region of the gamma subunit of cGMP phosphodiesterase with the GTP-bound alpha subunit of transducin. 890 Jan 74

The effects of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on a methionine-enkephalin (Met-E)-induced K+ current recorded from B-cluster neurons in Aplysia cerebral ganglion were investigated with voltage-clamp and pressure ejection techniques. Bath-applied SNP (10-25 microM) reduced the Met-E-induced K+ current in the neurons without affecting the resting membrane conductance and holding current. The inhibitory effects of SNP were reversible. Pretreatment with methylene blue (10 microM), a non-specific inhibitor of guanylate cyclase, and hemoglobin (50 microM), a NO scavenger, decreased the SNP-induced inhibition of the Met-E-induced current. Intracellular injection of 1 mM guanosine 3',5'-cyclic monophosphate (cGMP) or bath-applied 3-isobutyl-1-methylxanthine (IBMX; 50 microM), a nonspecific phosphodiesterase inhibitor, inhibited the Met-E-induced current. Furthermore, 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM), a more specific inhibitor of NO-stimulated guanylate cyclase, decreased the SNP-induced inhibition of the Met-E-induced current. These results suggest that SNP induces suppression of the Met-E-induced K+ current recorded from B-cluster neurons of Aplysia cerebral ganglion via stimulation of cGMP formation.
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PMID:Inhibition of the Met-enkephalin-induced K+ current in B-cluster neurons of Aplysia by nitric oxide donor. 897 6

A novel calcium-binding protein (EhCaBP) has been recently identified and characterized from the protozoan parasite Entamoeba histolytica. In order to decipher the function of this protein, a few basic properties were investigated and compared with the ubiquitous Ca(2+)-signal transducing protein calmodulin (CaM). Indirect immunofluorescence and immunoprecipitation analyses using specific antibodies against EhCaBP suggest that it is a soluble cytoplasmic protein with no major post-translational modification. EhCaBP did not stimulate cAMP-phosphodiesterase activity, differentiating it from all known CaMs. Affinity chromatography of [35S]methionine-labelled proteins of E. histolytica trophozoites using EhCaBP-sepharose column showed Ca(2+)-dependent binding of a group of proteins. Radiolabelled proteins from the same extract also bound to CaM-sepharose. However, the proteins bound to the two columns were different as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At least one of the EhCaBP-binding proteins became phosphorylated as revealed by in vivo phosphorylation analysis. The binding-proteins could not be detected in E. invadens (a species that is pathogenic in reptiles) and E. moshkovskii (which is found in the human gut but is not pathogenic), two species in which EhCaBP-like protein has not been found. Two distinct Ca(2+)-dependent protein kinases, which get activated by EhCaBP and CaM respectively, were detected in E. histolytica. These kinases require different levels of Ca2+ for their maximal activities. Affinity chromatography also showed the binding of protein kinase(s) to EhCaBP in a Ca(2+)-dependent manner. Our data suggest that there may be novel Ca(2+)-signal transduction pathway in E. histolytica mediated by EhCaBP.
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PMID:Characterization of EhCaBP, a calcium-binding protein of Entamoeba histolytica and its binding proteins. 904 22

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


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