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)

1. Neurons with a receptor responded to FMRFamide (Phe-Met-Arg-Phe-NH2) were identified in the ganglion of Aplysia kurodai. Ionic mechanism and channel gating system of the FMRFamide-induced responses were investigated by current clamp and voltage clamp methods. 2. The reversal potential of FMRFamide-induced response exactly coincided with the equilibrium potential for K+. This proved that the response was produced by a specific increase in membrane permeability toward K+, exclusively. 3. The FMRFamide-induced response was not affected by the inhibitors for Ca2(+)-activated K(+)-current, i.e., TEA, apamin, and EGTA. This excluded a possibility that FMRFamide-activated K(+)-channel is a Ca2(+)-activated K(+)-channel. 4. Intracellular injection of pertussis-toxin (PTX) caused no change in either resting potential or conductance, but it irreversibly blocked the FMRFamide-induced outward current within 30 min. Similarly applied cholera toxin (CTX) showed no effect on the FMRF-amide response. 5. Intracellular application of guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) caused no effect on either resting potential or conductance, but it blocked the FMRFamide-induced K(+)-current within 3 min. 6. Intracellular application of guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S) alone induced a slowly developing, irreversible outward current associated with an increase in membrane conductance. However, repetitive applications of FMRFamide immediately after the start of GTP gamma S application markedly facilitated the effect of GTP gamma S on the resting membrane. 7. Intracellular application of either adenylate cyclase inhibitor (3'-deoxyadenosine) or A-kinase inhibitor (H-8) did not affect the FMRFamide-induced response. 8. It was concluded that the FMRFamide-induced K(+)-current is mediated by PTX-sensitive GTP-binding protein Gi, Go or Gk. It was also suggested that the FMRFamide-induced response is produced independently of the changes in intracellular Ca2+ or cyclic AMP.
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PMID:[The gating mechanism of K(+)-channels coupled to the FMRFamide receptor in the ganglion cells of Aplysia]. 255 80

The regulatory subunit of the cAMP-dependent protein kinase expressed in clones isolated by immunoscreening of a lambda gt11 cDNA library from Dictyostelium discoideum exhibits high affinity for cAMP [Mutzel et al., Proc. Natl. Acad. Sci. USA 84 (1987) 6-10]. Based on this property, we have developed a screening procedure to detect in situ cAMP-binding activity directly on phage plaques transferred to nitrocellulose filters. Highly radioactive cAMP was synthesized using [alpha-32P]ATP at 3000 Ci/mmol as the substrate of purified adenylate cyclase from Bordetella pertussis. Filter replicas of the library plated at 3 X 10(4) pfu/dish, were incubated in the presence of 2 nM [32P]cAMP and then washed thoroughly. Three clones out of 1.2 X 10(5) were detected, all of which coded for the regulatory subunit, as judged by hybridization with a specific DNA probe. The cAMP binding to the purified clones was characterized in situ by displacement with specific analogues. The ability to displace labelled cAMP was in accord with the affinities of the analogues previously reported for the regulatory subunit of the Dictyostelium cAMP-dependent protein kinase. We are able to detect fmol levels of regulatory subunit contained in phage plaques and therefore the method could be used to screen libraries from other organisms for proteins exhibiting high affinities for cyclic nucleotides.
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PMID:Gene isolation by direct in situ cAMP binding. 282 98

GH exerts a number of metabolic effects on adipose tissue. Depending on the circumstances, it may increase or decrease glucose metabolism and lipolysis. These effects appear to be mediated by a single class of receptors, which bind GH with high affinity. Incubation of isolated rat adipocytes with a variety of lipolytic agents, including catecholamines, forskolin, or (Bu)2cAMP, decreased the specific binding of [125I]human (h) GH within 10 min. In the presence of 10 microM forskolin, GH binding declined to less than 20% of the control value within 50 min. Cholera and pertussis toxins, which increase cAMP secondary to ADP ribosylation of guanine nucleotide-binding proteins associated with hormone receptors, also decreased the binding of GH. None of these agents affected the rate of loss of cell-associated 125I when added to cells that had previously equilibrated with [125I]hGH. The inhibitory effects of forskolin and (Bu)2cAMP were at least as great when binding was measured in the presence of the protease inhibitor leupeptin, suggesting that increased rates of internalization and processing of bound hormone could not account for the decline in binding. Scatchard plots of data obtained in the presence of forskolin or (Bu)2cAMP were linear and parallel to control plots, indicating that the decline in binding could be accounted for by a decrease in the number of binding sites, with no change in affinity. To determine whether phosphorylation affected binding to receptors already present in the membrane or modified the turnover of receptors, we studied adipocyte ghosts, whose cellular apparatus for receptor turnover is disrupted. Incubation of adipocyte ghosts with cAMP-dependent protein kinase decreased the binding of [125I]hGH by 25%. The data suggest that cAMP-dependent phosphorylation of the GH receptor or a closely associated membrane protein renders the receptor incapable of binding GH.
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PMID:Adenosine 3',5'-monophosphate-dependent loss of growth hormone binding in rat adipocytes. 283 58

In the present work the molecular mechanisms of glucagon-induced desensitization of adenylate cyclase in cultured Sertoli cells have been studied in both whole cells and in a cell-free system. 1) Pretreatment of both whole Sertoli cells and membranes with glucagon induces a time- and dose-dependent desensitization of adenylate cyclase response that is primarily homologous and similar in the two systems. The component of heterologous desensitization, estimated by the reduced responsiveness to other hormones and NaF, accounted for only 12-20% loss of glucagon-responsive adenylate cyclase activity (P less than 0.01). 2) Glucagon-induced desensitization is ATP-dependent. Half maximal desensitization was achieved between 0.1 and 0.2 mM of ATP. 3) The typical time lag in the maximal activation of adenylate cyclase by GMPP(NH)P in the absence of hormone reappeared upon desensitization in spite of the presence of glucagon. The lag, however, was eliminated by FSH, showing that the homologous desensitization is due to a receptor-specific alteration. 4) The heterologous component of glucagon-induced desensitization is largely cAMP/protein kinase dependent. cAMP/protein kinase-induced desensitization was heterologous and caused approximately 30% loss of both hormonal and fluoride-stimulated enzyme activity. 5) Glucagon-induced desensitization is not due to altered activity of Ni since it proceeded equally well in membranes of cells pretreated with pertussis toxin (100 ng/ml) which eliminates Ni-mediated effects. It is concluded that glucagon induces both homologous and heterologous desensitization of the Sertoli cell adenylate cyclase. The locus of homologous desensitization appears to be at the level of the receptor and most probably involves a cAMP-independent phosphorylation reaction, whereas the heterologous desensitization appears to be cAMP-mediated and at least involves impaired functional activity of the Ns component.
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PMID:Mechanisms of glucagon-induced homologous and heterologous desensitization of adenylate cyclase in membranes and whole Sertoli cells of the rat. 284 Feb 61

Although dopamine inhibits PRL release from the normal anterior pituitary lactotroph, a conclusive demonstration of the mechanisms involved in this response has been impeded by the presence of other cell types in the anterior pituitary. To circumvent this problem, we have isolated a clonal cell line, designated MMQ, from the 7315a rat pituitary tumor. The MMQ cell is an exemplary model for our use because it only secretes PRL. Our studies show that dopamine inhibits secretagogue-induced PRL release from these cells. In addition, dopamine decreases the intracellular cAMP concentration in MMQ cells that have been exposed to forskolin, cholera toxin, or vasoactive intestinal polypeptide, each a stimulator of cAMP generation. This inhibition is, in turn, reversed by the dopamine antagonist haloperidol and by pertussis toxin, an inactivator of the GTP-binding coupling protein. Dopamine also decreases the uptake and fractional efflux of 45Ca2+ by MMQ cells that have been exposed to the calcium channel activator maitotoxin. It seems, therefore, that dopamine decreases PRL release from MMQ cells at least in part by decreasing intracellular cAMP levels and calcium uptake. In additional experiments, we have found that MMQ cells are responsive to somatostatin, estrogen, progesterone, and acetylcholine, but not to TRH, angiotensin II, neurotensin, or bombesin. Furthermore, these cells possess a functional protein kinase-C system, as evidenced by the increase in PRL release and decrease in stimulated intracellular cAMP levels that occur in response to treatment with phorbol diesters. We suggest that the MMQ cell line will prove a useful model system for study of the biochemical effects of dopamine and other factors that modify PRL release.
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PMID:Characterization of the MMQ cell, a prolactin-secreting clonal cell line that is responsive to dopamine. 284 8

The regulation of the cytosolic calcium concentration was investigated in freshly isolated adult bovine tracheal smooth muscle cells using fura 2. These cells contain 1.1 and 1.8 pmol of cGMP kinase and cAMP kinase per mg protein, respectively. Carbachol, histamine, serotonin, isoproterenol, and salbutamol increased the cytosolic calcium in a dose-dependent manner from 79 nM to about 650 nM. Preincubation of these cells for 20 min with isoproterenol, forskolin, 8-Br-cAMP and 8-(4-Cl-phenyl)thio-cAMP did not lower carbachol-induced increases in cytosolic calcium concentration, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the atrionatriuretic factor, isobutylmethylxanthine, and 8-Br-cGMP lowered cytosolic calcium. The active fragment of cGMP kinase, but not the catalytic subunit of cAMP kinase lowered carbachol-induced calcium levels. Carbachol released calcium from intracellular stores and increased calcium influx from the extracellular space. The influx was inhibited by preincubation with the calcium channel blockers nitrendipine or gallopamil. Both carbachol-stimulated pathways were suppressed by 8-Br-cGMP. Isoproterenol increased only the influx of calcium from the outside by a channel which was blocked by calcium channel blockers or 8-Br-cGMP. Forskolin and 8-Br-cAMP lowered carbachol- and isoproterenol-stimulated increases in calcium when added shortly before or after the addition of the agonist. In addition, isoproterenol decreased carbachol-stimulated calcium levels when added 10 s after carbachol. The calcium stimulatory effect of isoproterenol was abolished by preincubation of the cells with pertussis toxin or cholera toxin. These results show (a) that the beta 2-adrenoceptor couples in isolated tracheal smooth muscle cells to a dihydropyridine- and pertussis toxin-sensitive calcium channel; (b) that the same channel is opened by carbachol; (c) that cGMP kinase is very effective in decreasing elevated cytosolic calcium concentrations, whereas cAMP-dependent protein kinase has a variable effect on stimulated cytosolic calcium levels.
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PMID:Regulation of cytosolic calcium by cAMP and cGMP in freshly isolated smooth muscle cells from bovine trachea. 284 48

Preincubation of human neutrophils with recombinant tumor necrosis factor alpha has previously been shown by us to enhance superoxide production of neutrophils in response to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine, and the phorbol ester, phorbol myristate acetate. In this study, we further investigate the biochemical basis for this enhancement. We found that in neutrophils, TNF by itself does not induce: (1) an influx of sodium, (2) an alteration in activity or translocation of the calcium and phospholipid dependent protein kinase (C-kinase), or (3) a release of arachidonic acid from preloaded cells. TNF did, however, induce a time- and concentration-dependent increase in the phosphorylation of several neutrophil proteins, with the most dramatic concentration dependent increase in a 64,000 Da protein. Finally, the enhancement of O2 production by pretreatment of neutrophils with TNF was found to be independent of a pertussis toxin-sensitive guanine nucleotide regulatory protein.
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PMID:Biochemical mechanisms involved in the priming of neutrophils by tumor necrosis factor. 284 26

Addition of bombesin in the presence of either forskolin or cholera toxin caused a marked (4-6 fold) enhancement of cAMP accumulation in Swiss 3T3 cells. This effect was time and concentration dependent, induced by various bombesin-like peptides and blocked by a bombesin antagonist. Enhancement of cAMP accumulation by bombesin was diminished by chronic pretreatment with phorbol dibutyrate implicating the involvement of protein kinase C in the activation. Pretreatment with pertussis toxin, which uncouples protein kinase C activation from cAMP accumulation (Proc. Natl. Acad. Sci. U.S.A., 84:2282, 1987) also inhibited bombesin enhancement of cAMP. Bombesin was also shown to release E type prostaglandins into the medium, an effect which was abolished by the cyclooxygenase inhibitor indomethacin. Low concentrations (100 nM) of indomethacin partially inhibited the accumulation of cAMP by bombesin in the presence of forskolin indicating that the release of E type prostaglandins into the medium is also involved in the accumulation of cAMP by bombesin. The additive nature of PBt2-mediated down-regulation and treatment with indomethacin suggests that activation of protein kinase C and the release of E type prostaglandins provide two distinct pathways involved in the enhancement of cAMP accumulation by bombesin. Finally, bombesin in the presence of forskolin stimulated the phosphorylation of the intermediate filament component vimentin, identified in the accompanying paper as a substrate for a cAMP dependent protein kinase in intact Swiss 3T3 cells.
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PMID:Bombesin enhancement of cAMP accumulation in Swiss 3T3 cells: evidence of a dual mechanism of action. 284 40

Nanomolar concentrations of synthetic peptides corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase were found to inhibit calmodulin activation of seven well-characterized calmodulin-dependent enzymes: brain 61 kDa cyclic nucleotide phosphodiesterase, brain adenylate cyclase, Bordetella pertussis adenylate cyclase, red blood cell membrane Ca++-pump ATPase, brain calmodulin-dependent protein phosphatase (calcineurin), skeletal muscle phosphorylase b kinase, and brain multifunctional Ca++ (calmodulin)-dependent protein kinase. Inhibition could be entirely overcome by the addition of excess calmodulin. Thus, the myosin light chain kinase peptides used in this study may be useful antagonists for studying calmodulin-dependent enzymes and processes.
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PMID:Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulin-dependent enzymes. 290 35

We prepared and characterized subcellular membrane fractions from the CNS of Aplysia californica that are enriched in isolated nerve terminals (synaptosomes). Ganglia were homogenized in 1.1 M sucrose and fractionated on a 2-step sucrose gradient, yielding 50 micrograms protein/animal in the synaptosomal fraction (P3), which was enriched 3-fold in plasma membrane as compared with the initial homogenate. Quantitative morphometry of electron micrographs revealed that P3 contained 25% intact synaptosomes, a 5-fold enrichment over the homogenate. Although fractionation on a 5-step sucrose gradient reduced the yield of protein in the synaptosomal fraction to 40 micrograms/animal, this fraction (the 0.35 M/0.75 M interface) was more enriched in plasma membrane than P3 and was less contaminated by lysosomes and free mitochondria. By electron microscopy, the 0.35 M/0.75 M interface contained up to 50% synaptosomes. Synaptosomal fractions contained cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinase activities and were enriched in a Mr 40,000 pertussis toxin substrate, Gi/o. In the accompanying paper, we show that these synaptosomes retain the ability to release transmitters.
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PMID:Aplysia synaptosomes. I. Preparation and biochemical and morphological characterization of subcellular membrane fractions. 291 12


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