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)

An assay for cyclic AMP is described which takes advantage of the high affinity of the dissociated receptor moiety of cyclic AMP-dependent protein kinase I for the nucleotide. The kinase is kept dissociated by salt (800 mM-NaCl/30mM-EDTA). In the presence of a simply prepared heat-stable protein fraction the binding reagent is stable for the time needed to reach equilibrium of binding. A simple procedure [precipitation with poly-(ethylene glycol) followed by DEAE-cellulose chromatography] is described for the separation of protein kinase I from other binding proteins for cyclic AMP in rabbit skeletal muscle. The sensitivity, precision, reproducibility and specificity of the assay compared favourably with those of other cyclic AMP assays. The main advantage of the present assay is its resistance towards non-specific interference from a number of salts, tissue-culture media and substances found in crude tissue extracts. The reliability of cyclic AMP measurement directly in crude tissue extracts was ensured by removal of the assayable cyclic AMP with cyclic nucleotide phosphodiesterase digestion or adsorption with antibody against cyclic AMP, by comparison with measurement in tissue extracts purified by chromatography on QAE-Sephadex or sequentially on Dowex 50, and aluminium oxide as well as by dilution and recovery experiments.
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PMID:Measurement of adenosine 3':5'-cyclic monophosphate by competitive binding to salt-dissociated protein kinase. 21 54

Measurements of tissue cyclic AMP (cAMP) concentration, the activity of cAMP-dependent protein kinase and the level of the enzyme's thermostable, macromolecular inhibitor were made on preparations of rat epididymal fat pad from animals fed high fat or high carbohydrate diets. The cAMP concentration from rats adapted to a high lard diet for 14-15 days was 153 +/- 17.8 pmoles/mg protein as opposed to 76 +/- 6.0 found with high glucose diet. No significant difference in total cAMP-dependent protein kinase activity was observed among rats fed high glucose, high lard or laboratory chow, although the enzyme's activity ratio (-cAMP)(+cAMP) was significantly elevated with lard feeding (0.49 +/- 0.02) as opposed to glucose feeding (0.43 +/- 0.01). Crude preparations from lard and glucose fed animals were equivalent in inhibitory activity when tested with enzyme from chow fed animals. Agarose column chromatography separated holoenzyme and C subunit forms of the protein kinase when 500 mM NaCl was present in the elution buffer. Absence of the salt allowed subunit reassociation to occur. Direct addition of NaCl greater than or equal to 75 mM significantly inhibited protein kinase activity. The results indicate that the adipose tissue of rats fed a high lard diet has a higher concentration of cAMP and an increased protein kinase activity ratio than tissue from rats fed a fat free, high glucose diet. Total cAMP-dependent protein kinase activity and the level of a thermostable macromolecular inhibitor remained unchanged.
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PMID:The concentration of cyclic AMP and the activity of cyclic AMP dependent protein kinase and an inhibitor in the adipose tissue of rats fed lard or glucose diets. 21 69

A photoaffinity labeling method was used to characterize and compare cyclic nucleotide-binding proteins of bovine liver cytosol with binding proteins of the nucleus. After photoaffinity labeling of cytosol with 8-azido cyclic [(32)P]AMP, autoradiographs of sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed two major labeled proteins of 47,000 and 52,000-55,000 daltons. DEAE-cellulose column-derived fractions suggested that the larger protein was the regulatory subunit of peak II cyclic AMP-dependent protein kinase and the smaller protein was the regulatory subunit of peak I kinase. The smaller protein was largely present as the free regulatory subunit. The two binding proteins differed in their ability to bind cyclic GMP. Binding to both proteins was abolished by excess unlabeled cyclic AMP but not by 5'-AMP. Photoaffinity labeling of a 0.14 M salt extract of nuclei and a nonhistone chromosomal protein preparation revealed two major binding proteins with the same molecular weight and competition profiles as those of the cytosol. Detergent-washed nuclei gave similar results. Several minor binding proteins were observed in both cytosol and nucleus. One protein (36,000 daltons) was unique to the nucleus and had low affinity for 8-azido cyclic AMP. Photoaffinity labeling with cyclic [(3)H]GMP revealed a cytosol protein, absent from the nucleus, of 31,000 daltons and the ligand was competed for by both cyclic GMP and 5'-GMP. These studies suggest that the major specific cyclic AMP-binding proteins of bovine liver are the type I and type II regulatory subunits of cyclic AMP-dependent protein kinase and are present in both nucleus and cytoplasm.
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PMID:Cyclic nucleotide-binding proteins detected by photoaffinity labeling in nucleus and cytoplasm of bovine liver. 21 81

Chinese hamster ovary cells exhibit several characteristic morphological and physiological responses upon treatment with agents which increase the intracellular level of adenosine 3':5'-phosphate (cyclic AMP). To better understand the mechanism of these cyclic AMP-mediated responses, we separated two cyclic AMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) (protein kinase I and protein kinase II) from the cytosol of Chinese hamster ovary cells by DEAE-cellulose chromatography and studied their properties. Protein kinase I is eluted at a lower salt concentration than protein kinase II and is stimulable to 10 times its basal catalytic activity, while protein kinase II is stimulable only 2-fold. Both kinases are completely dissociated by cyclic AMP and inhibited by specific cyclic AMP-dependent protein kinase inhibitor. They have similar Km values for magnesium (approximately 1 mM), cyclic AMP (approximately 60 nM), and ATP (approximately 0.1 mM), and the dissociation constant (Kdis) for cyclic AMP (approximately 13 nM) is the same for both enzymes. However, they appear to have different substrate preferences and cyclic AMP-binding properties in that cyclic AMP bound to protein kinase II exchanges readily with free cyclic AMP, while that bound to protein kinase I is not exchangeable. The native enzymes have different sedimentation coefficients (6.4 S for protein kinase I and 4.8 S for protein kinase II), whereas those of the activated enzymes are the same (2.9--3.0 S). It appears that the two cyclic AMP-dependent protein kinases which differ from each other in their regulatory subunits may play different roles in the mediation of cyclic AMP action in Chinese hamster ovary cells.
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PMID:Characterization of two adenosine 3':5'-phosphate-dependent protein kinase species from Chinese hamster ovary cells. 21 11

Cytosol of mature estrous rabbit follicles contains a single species of protein kinase, protein kinase 3, which can be classified as a type II cAMP-dependent protein kinase. Cytosol of functional rabbit corpora lutea (CL) contains, in addition to protein kinase 3, a second species of kinase activity, protein kinase 2, which can be classified as a type I cAMP-dependent protein kinase. These conclusions are based upon the relative dissociation and reassociation characteristics of the two holoenzymes in the presence and absence of 0.5 M NaCl after in vitro dissociation by cAMP, upon the effect of MgATP on salt- and basic protein-induced dissociation, and upon their relative elution from DEAE-cellulose. Protein kinase 3 in mature estrous rabbit follicles was rapidly activated after an iv injection of hCG. The activation was demonstrated by an increase of the protein kinase activity ratio as well as by the appearance of the free catalytic subunit of protein kinase upon Sephadex gel filtration. Maximal activation occurred within 10 min of in vivo hormone administration and required ovulatory doses of hormones with LH-like activity. Neither PRL, ACTH, epinephrine, nor a highly purified preparation of FSH promoted activation of the follicular protein kinase 3. Demonstration of protein kinase activation in follicles was achieved in the presence of 0.5 M NaCl in the homogenization media. After an iv injection of hCG, a partial activation of luteal protein kinases 2 and 3 was demonstrated, as reflected by the increase of the protein kinase activity ratio. These results implicate an important role for cAMP-dependent protein kinase 3 in LH action in rabbit ovarian follicles and for cAMP-dependent protein kinases 2 and 3 in LH action in rabbit CL.
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PMID:Rabbit ovarian protein kinases. III. Gonadotrophin-induced activation of soluble adenosine 3',5'-monophosphate-dependent protein kinases. 21 48

Phosphorylation of H1 histones by cyclic AMP-dependent protein kinase may be an important transcriptional control mechanism. We have used affinity chromatography to examine the effect of phosphorylation by this enzyme on the DNA-binding properties of calf thymus H1 histones and two highly basic H1 homologues from condensed and transcriptionally silent nuclei: duck erythrocyte H5 and Strongylocentrotus purpuratus sperm H1. Without in vitro phosphorylation, all three histones were eluted from native DNA-Sephadex G-25 columns at salt concentrations which closely resembled those required to extract these histones from nuclei or chromatin. When a small portion of radioactively phosphorylated histone was chromatographed with untreated carrier histone, the phosphorylated species was consistently eluted from the DNA column at slightly lower salt concentrations than the main histone peak. Rechromatography experiments showed that in vitro phosphorylation of H1 can shift its elution position to lower salt concentrations.
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PMID:Phosphorlyation of H1 and H5 histones by cyclic AMP-dependent protein kinase reduces DNA binding. 22 45

When myofibrils from rat hearts were dissolved in concentrated salt solutions and reprecipitated by dilution, they contained both protein kinase (partly cyclic 3':5'-AMP-dependent) and protein phosphatase activities. Troponin-I was the major protein to be phosphorylated by the endogenous myofibril-associated kinase and by added protein kinase. Approximately 1 mole of phosphate per mole of troponin-I was incorporated from radioactive ATP, but the extent of troponin-I phosphorylation could be varied experimentally. An inverse correlation was found between protein phosphorylation and the maximum Ca2+-stimulated myofibrillar Mg2+-ATPase activity, while the amout of calcium required for half-maximum activation was proportional to the extent of protein phosphorylation. The changes in Mg2+-ATPase activity produced in vitro by protein phosphorylation were reproduced in isolated perfused rat hearts treated for short periods with L-noradrenaline (10(-6)M). The changes in myofibrillar function brought about as the result of the phosphorlyation by cAMP-dependent protein kinase suggest that the contractile response is desensitized in order to cope with the rise in intracellular Ca2+ which results from the action of catecholamines on cardiac ventricular cells.
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PMID:Cardiac myofibrillar phosphorylation and adenosine triphosphatase activity. 22 75

Highly purified Sendai virus contained a protein kinase activity which atatlysed the phosphorylation of endogenous polypeptides or exogenous protamine sulphate. The virus contained very low levels of phosphoprotein phosphatase activity. Polyacrylamide gel analysis of the reaction product indicated that the phosphorylation was specific for certain polypeptides and varied according to whether the virus was grown in eggs or in tissue culture. This variation was partially associated with the difference in the polypeptide pattern that occurred when the virus was grown in eggs or in tissue culture. Characterization of these phosphoproteins demonstrated that the phosphate was incorporated predominantly in a phosphoester linkage with theonine residues. Using a detergent and high salt solubilization procedure, the protein kinase activity was found associated within glycoprotein free virus particles but not with the nucleocapsid-associated polypeptides. In vivo phosphorylation occurred when Sendai virus was grown in eggs or in tissue culture with [32P] and the phosphorylated polypeptides were similar to those of the protein kinase reaction product. Phosphorylation could also be detected in the infected cell and could occur once the virus particle polypeptides were being synthesized. The non-structural polypeptides were not phosphorylated.
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PMID:The phosphorylation of sendai virus proteins by a virus particle-associated protein kinase. 23 97

Despite the finding that the hemin-controlled translational inhibitor in reticulocyte lysates is a cyclic AMP-independent protein kinase that phosphorylates the small subunit of the initiation factor eIF-2, the mechanism of inhibition of translation remained unexplained. Whereas treatment of hemin-containing lysates with inhibitor in the presence of ATP inhibited translation, the same treatment of highly purified eIF-2 did not affect its ability to form a ternary complex with initiator Met-tRNA and GTP or a 40S initiation complex. We have isolated from ribosomal salt washes a protein (eIF-2 stimulating protein) that enhances the capacity of unphosphorylated eIF-2 to form ternary or 40S initiation complexes but has no effect on the phosphorylated factor. At low concentrations, eIF-2 is virtually inactive without this stimulating protein. Therefore, the translational inhibitor acts by converting eIF-2 to a form that is not stimulated by the stimulating protein.
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PMID:Mode of action of the hemin-controlled inhibitor of protein synthesis. 27 39

The phosphorylation of purified protein synthesis factors catalyzed by protein kinase preparations isolated from interferon-treated human amnion cells was examined. Ribosomal salt-wash fractions prepared from interferon-treated human cells contained a protein kinase that catalyzed the [gamma-(32)P]ATP-mediated phosphorylation of the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2alpha); this kinase activity was significantly enhanced in interferon-treated as compared to untreated cells. The tryptic [(32)P]phosphopeptide pattern obtained for eIF-2alpha phosphorylated by the interferon-mediated human kinase was indistinguishable from the pattern obtained for eIF-2alpha phosphorylated by the hemin-regulated rabbit reticulocyte kinase when analyzed by thin-layer chromatography with three different solvent systems and by high-voltage electrophoresis. O-[(32)P]Phosphoserine was liberated by partial acid hydrolysis from eIF-2alpha phosphorylated by either the human or the rabbit kinase. In addition to the phosphorylation of eIF-2alpha, interferon treatment of human cells enhanced the phosphorylation of two additional ribosome-associated proteins designated P(1) and P(f). The major phosphoester linkage observed for the human, as well as murine, phosphoprotein P(1) was O-phosphoserine. The interferon-mediated phosphorylation of both eIF-2alpha and protein P(1) was dependent upon the presence of RNA with double-stranded character; P(f) phosphorylation was not affected by double-stranded RNA. These results suggest that the interferon-mediated ribosome-associated human protein kinase catalyzes the phosphorylation of eIF-2alpha in a site-specific manner that is apparently identical with the reaction catalyzed by the hemin-regulated rabbit reticulocyte kinase; hence, the phosphorylation of eIF-2 may play a role in regulating the initiation of translation in interferon-treated cells.
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PMID:Mechanism of interferon action: phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosome-associated kinase processing site specificity similar to hemin-regulated rabbit reticulocyte kinase. 28 84


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