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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4-5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50-2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose-response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [(3)H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [(3)H]AMP, the radioactivity associated with the cells increased almost linearly up to 250mum-cyclic [(3)H]AMP concentration in the incubation medium. The (3)H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.
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PMID:Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3':5'-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells. 22 66

Evidence is presented for the presence of multiple cyclic AMP binding components in the plasma membrane and cytosol fractions of porcine renal cortex and medulla. N6-(Ethyl-2-diazomalonyl)-3',5'-adenosine monophosphate, a photoaffinity label for cyclic AMP binding sites, exhibits non-covalent binding characteristics similar to cyclic AMP in membrane and soluble fractions. Binding data for either compound to the plasma membrane fraction yields biphasic Scatchard plots while triphasic plots are obtained with the dialyzed cytosol. When covalently labeled fractions are separated on SDS-polyacrylamide gel electrophoresis, the cyclic AMP photoaffinity label is found on 49 000 and 130 000 dalton components in each kidney fraction. DEAE-cellulose and gel filtration chromatography of the labeled cortical cytosol fraction establishes that the three components suggested by the binding data correspond to two 49 000 dalton species and a 130 000 component. The 49 000 species have higher affinities for cyclic AMP than the 130 000 component (Ka(1) = 2.0 . 10(9), Ka(2) = 1.7 . 10(8), Ka(3) = 1.0 . 10(7)). The 49 000 components are associated with protein kinase activity while the 130 000 component does not exhibit protein kinase, adenosine deaminase, or cyclic nucleotide phosphodiesterase activity. Immunologic results and effects of phosphorylation and cyclic GMP on cyclic AMP binding further suggest that the 49 000 components are regulatory subunits of cyclic AMP-dependent protein kinases. Cyclic AMP binding to the 130 000 component is markedly inhibited by adenosine and adenine nucleotides, but not cyclic GMP. Thus, this component may reflect an aspect of adenosine control or metabolism which may or may not be a cyclic AMP-related cellular function.
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PMID:Photoaffinity labeling of three renal cyclic 3',5'-adenosine monophosphate-binding proteins. 22 50

A number of 8- and N6-SUBSTITUTED DERIVATIVES OF CYCLIC ADENOSINE 3':5'-MONOPHOSPHATE-DEPENDENT PROTEIN KINASE, AND AS SUBSTRATES FOR RAT LIVER CYCLIC NUCLEOTIDE PHOSPHODIESTERASE. All of the analogs tested were able to induce the transaminase. The induction by the analogs was shown to be the result of an actual increase in the amount of enzyme, and the mechanism of induction was an increase in the rate of synthesis of the transaminase. The induced enzyme appeared to be immunologically similar to the non-induced enzyme. A good correlation was found to exist between the dose that produced 50% of maximal induction and a combination of the activation constant for cyclic adenosine 3':5'-monophosphate-dependent protein kinase by the analog and its susceptibility to hydrolysis by cyclic nucleotide phosphodiesterase. These data suggest that the phosphorylation of some site is involved in the mechanism by which cyclic adenosine 3':5'-monophosphate affects the rate of synthesis of tyrosine aminotransferase.
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PMID:Induction of hepatic tyrosine aminotransferase in vivo by derivatives of cyclic adenosine 3':5'-monophosphate. 23 51

The control of protein synthesis by hemin in rabbit reticulocytes is mediated by the formation of a high molecular weight protein inhibitor of polypeptide chain initiation, termed the hemin-controlled translational repressor, from a presynthesized prorepressor. The prorepressor, purified approx. 600-fold, was used to study the mechanism of hemin-controlled translational repressor formation. When the prorepressor is converted to the hemin-controlled translational repressor, either by prolonged warming in the absence of hemin or by incubation with N-ethylmaleimide for 5 min, and then incubated briefly with [gamma-32P]-ATP and Mg2+, a protein that migrates as a 100 000 molecular weight component on sodium dodecyl sulfate-polyacrylamide gels becomes phosphorylated. The extent of phosphorylation of this component is directly proportional to the amount of prorepressor converted to the hemin-controlled translational repressor. In addition, the 100 000 molecular weight protein is not labeled when phosphorylation is attempted with the prorepressor or prorepressor warmed in the presence of hemin, indicating that the protein kinase responsible is probably the hemin-controlled translational repressor. Since the 100 000 molecular protein copurifies with the prorepressor and since the phosphorylation reaction is very rapid (50% complete within 30 s at 34 degrees C), relatively insensitive to dilution, and behaves like an intramolecular reaction, the data suggest that the hemin-controlled translational repressor, once activated, may autophosphorylate a 100 000 molecular weight subunit of itself. Approx. 5 mol phosphate are incorporated per mol of 100 000 molecular weight protein, when the prorepressor is completely converted to the hemin-controlled translational repressor by N-ethylmaleimide. Neither the rate of conversion of prorepressor to the hemin-controlled translational repressor nor the subsequent phosphorylation of the 100 000 molecular weight protein is enhanced by cyclic AMP or reduced by incubation with 3':5'-cyclic nucleotide phosphodiesterase, indicating that cyclic AMP plays no role in hemin-controlled translational repressor formation.
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PMID:Control of protein synthesis by hemin. An association between the formation of the hemin-controlled translational repressor and the phosphorylation of a 100 000 molecular weight protein. 71 20

Calmodulin is the major intracellular Ca(2+)-binding protein, providing Ca(2+)-dependent regulation of numerous intracellular enzymes. The phosphorylation of calmodulin may provide an additional mechanism for modulating its function as a signal transducer. Phosphocalmodulin has been identified in tissues and cells, and calmodulin is phosphorylated both in vitro and in intact cells by various enzymes. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreases its ability to activate both myosin-light-chain kinase and cyclic nucleotide phosphodiesterase. For myosin-light-chain kinase the primary effect is an inhibition of the Vmax. of the reaction, with no apparent change in the concentration at which half-maximal velocity is attained (K0.5) for either Ca2+ or calmodulin. In contrast, for phosphodiesterase, phosphorylation of calmodulin significantly increases the K0.5 for calmodulin without noticeably altering the Vmax. or the K0.5 for Ca2+. The higher the stoichiometry of phosphorylation of calmodulin, the greater the inhibition of calmodulin-stimulated activity for both enzymes. Therefore the phosphorylation of calmodulin by casein kinase II appears to provide a Ca(2+)-independent mechanism whereby calmodulin regulates at least two important target enzymes, myosin-light-chain kinase and cyclic nucleotide phosphodiesterase.
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PMID:Phosphorylation by casein kinase II alters the biological activity of calmodulin. 131 63

Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction. The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells. The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity. After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed. Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed. Further deletions within the amino terminus produced inactive proteins. Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs. Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE. However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine. Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity. In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme. Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins. These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.
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PMID:Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain. 132 38

Murine melanoma cells treated with the melanocyte-stimulating hormone (MSH) family of peptides undergo differentiation characterized by enhanced melanogenesis and altered morphology. These effects are mediated via the adenylate cyclase-cAMP pathway leading to activation of protein kinase A (PKA). We have discovered that inhibition of a post-translational modification of chromatin proteins, viz. poly(ADP-ribosylation), also induces melanogenesis and differentiation in these cells. A range of competitive inhibitors (benzamide and its derivatives) of the nuclear enzyme poly(ADP-ribose) polymerase (PADPRP; EC 2.4.2.30) was utilized, and their ability to induce melanogenesis reflected their potency as PADPRP inhibitors. These compounds induced melanogenesis at low doses (20 microM-2 mM) which did not affect cell growth or viability. Induction of melanogenesis was not attributable to inhibition of cyclic nucleotide phosphodiesterase by these compounds. MSH treatment caused a transient rise in cAMP levels (up to 200-fold by 5 min and returning to near basal levels by 5 h). It also stimulated PKA activity up to 5-fold, and the temporal kinetics of this activation mirrored the changes in cAMP levels. In comparison, the PADPRP inhibitors had no effect on either of these processes. These data constitute a novel demonstration of a cAMP-independent mechanism for the induction of melanoma cell differentiation, including melanogenesis.
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PMID:Murine melanoma cell differentiation and melanogenesis induced by poly(ADP-ribose) polymerase inhibitors. 132 52

Biotinylated derivatives of calmodulin (CaM) were prepared and their biological properties characterized by using enzyme assays, affinity and hydrophobic-interaction chromatography. Several N-hydroxysuccinimidobiotin derivatives [sulphosuccinimidobiotin (sulpho-NHS) and sulphosuccinimido-6-(biotinamido)hexanoate (BNHS-LC)] differing in spacer arm length were used to modify CaM. The shorter-spacer-arm CaM derivative (sulpho-CaM) activated CaM-dependent cyclic nucleotide phosphodiesterase and CaM-dependent protein kinase II; preincubation with avidin blocked its ability to activate these enzymes. The extended-spacer-arm derivative (BNHS-LC-CaM) activated CaM-dependent enzymes both in the presence and in the absence of avidin, suggesting that the longer spacer arm diminished steric effects from avidin preincubation. Other biotinylated CaM derivatives were prepared with biotinylated tyrosine and/or histidine residues (diazobenzoylbiocytin; DBB-CaM) or nucleophilic sites (photobiotin acetate; photo-CaM). These derivatives activated CaM-dependent enzymes in the presence and in the absence of avidin. Oriented affinity columns were constructed with covalently immobilized avidin complexed to each biotinylated CaM derivative. The chromatographic profiles obtained revealed that each column interacted with a specific subset of CaM-binding proteins. Elution profiles of biotinyl CaM derivatives on phenyl-Sepharose hydrophobic-interaction chromatography suggested that several derivatives displayed diminished binding to the matrix in the presence of Ca2+. Development and characterization of a series of biotinylated CaM molecules can be used to identify domains of CaM that interact with specific CaM-dependent enzymes.
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PMID:Preparation, characterization and biological properties of biotinylated derivatives of calmodulin. 164 21

Experiments have been performed to characterize guinea-pig peritoneal eosinophil cyclic nucleotide phosphodiesterase (PDE) activity and establish whether it is involved in regulating superoxide (.O2-) generation. Eosinophils were found to contain a predominantly membrane-bound cAMP PDE(s) (92.5 +/- 2.4% of total activity) which was resistant to solubilization with Triton X-100 (1%). This particulate PDE exhibited complex kinetics (Km = 1.3 and 31.4 microM) and was unaffected by cGMP (IC50 greater than 100 microM) or CaCl2 (2 mM) + calmodulin (10 units/mL). Little cGMP PDE activity was detected in either the soluble or particulate fractions. Inhibitors of the Ro-20-1724-inhibited (Type IV) cAMP PDE, namely Ro-20-1724 (IC50 = 0.92 +/- 0.43 microM), rolipram (IC50 = 0.20 +/- 0.04 microM) and denbufylline (IC50 = 0.20 +/- 0.01 microM), potently inhibited the particulate cAMP PDE, as did the non-selective inhibitors trequinsin (IC50 = 0.11 +/- 0.02 microM) and AH-21-132 (IC50 = 2.57 +/- 0.02 microM). Eosinophil cAMP PDE was resistant to SK&F 94120 (IC50 greater than 1000 microM), the cGMP-inhibited (Type III) cAMP PDE inhibitor, and the cGMP PDE (Type I) inhibitor, zaprinast, was only weakly active (IC50 = 35.33 +/- 10.74 microM). .O2- release from resting cells was potently inhibited by rolipram (IC50 = 0.05 +/- 0.03 microM) and denbufylline (IC50 = 0.06 +/- 0.04 microM) but surprisingly, in view of its potent cAMP PDE inhibitory activity, was only weakly decreased by trequinsin (IC50 = 8.0 +/- 2.7 microM). AH-21-132 (IC50 greater than 10 microM), SK&F 94120 (IC50 greater than 10 microM) and zaprinast (IC50 greater than 10 microM) were without effect. Rolipram and denbufylline alone exerted little effect on cAMP in intact cells but, in the presence of 10 microM isoprenaline, potently increased intracellular accumulation (EC50 = 0.45 +/- 0.16 and 0.28 +/- 0.08 microM, respectively). Trequinsin and AH-21-132 only weakly enhanced isoprenaline-stimulated cAMP accumulation. Although it induced a marked rise in cAMP only in the presence of isoprenaline, rolipram (50 microM) alone was able to increase the activity ratio of cAMP-dependent protein kinase from 0.24 to 0.84. The results suggest that Ro-20-1724-inhibited cAMP PDE plays a role in regulating eosinophil .O2- generation. The poor correlation between the PDE inhibitory actions of certain compounds and their effectiveness in elevating cAMP and inhibiting .O2- suggests the existence of a barrier impeding access to the enzyme.
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PMID:Characterization of guinea-pig eosinophil phosphodiesterase activity. Assessment of its involvement in regulating superoxide generation. 165 Oct 83

Knowledge about second messenger metabolizing enzymes in neuroglia is still rather fragmentary. Therefore, the aim of the present investigation was to localize adenylate cyclase, guanylate cyclase, cyclic nucleotide phosphodiesterase and protein kinase A in glial cells of the rat hippocampus and cerebellum. Enzyme histochemical and immunohistochemical methods were used to detect the enzymes at the light and electron microscopic level. Astroglial cells were found to contain all 4 enzymes. Especially the microvascular glial cell processes were reactive. Oligodendroglial cells were only stained for adenylate cyclase acticity. Intracellularly, microtubules and intracellular membranes were frequently stained. The results point to the regulation of glial cell metabolism and of transport processes by cyclic nucleotides.
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PMID:Second messenger enzymes in glial cells: a cytochemical point of view. 168 99


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