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 level of endogenous protein phosphorylation in non-histone chromosomal and ribosomal wash proteins is 7--10 times greater in SV40-transformed rat cells than in untransformed parental cells. Protein kinase activity in these proteins was fractionated by either phosphocellulose or DEAE-cellulose chromatography. One major and one minor component were detected in non-histone proteins and only one component in ribosomal wash proteins when the activity in each fraction was measured with an exogenous substrate, casein. These enzymes prefer casein to whole histone as substrate and are cyclic AMP-independent. The enzyme activity in a major peak of non-histone proteins and in ribosomal wash proteins measured with casein as substrate is 3 times greater in transformed cells than in untransformed cells, whereas pH optimum, cation requirements and apparent Km values for casein and ATP are identical or very similar in the two cell types. No significant phosphatase was detected in non-histone and ribosomal wash proteins from the two types of cell. The patterns of endogenous protein phosphorylation in these protein fractions analysed by gel electrophoresis are significantly different between these cells. These results suggest that the high level of endogenous protein phosphorylation in non-histone and ribosomal wash proteins from SV40-transformed cells is caused mainly by the increased activity of protein kinase and the nature of protein substrates.
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PMID:Protein kinases and their protein substrates associated with chromatin and ribosomes in SV40-transformed rat cells. 21 21

Bovine thyroid cyclic AMP-dependent protein kinase was purified by DEAE-Sephadex and Sephadex G-200 chromatography. This preparation showed a 240-fold increase in specific activity over the initial 20,000 x g supernatant with histone as substrate and 1 micronM cyclic AMP in the assay mixture. In the presence of 2.5 X 10(-5)M L-triiodothyronine (T3), protein kinase activity was significantly reduced; 50% inhibition was achieved at 1 X 10(-4) M. Tests of diverse thyroid hormone analogs showed that T3 and its derivatives were more potent inhibitors than T4 and its derivatives which, in turn, were more potent than thyronine or diiodothyronine. Mono- and diiodotyrosine, tyrosine, and iodide were without effect. Triiodothyronine did not inhibit kidney, spleen, or lung protein kinase activity. The magnitude of the inhibition was the same whether or not cyclic AMP (1 micronM) was present in the incubation mixture, suggesting an effect on the catalytic, rather than the regulatory subunit of the enzyme. The inhibition of protein kinase by thyroid hormone was not influenced by Mg++ concentration but was overcome in a competitive manner by increasing ATP concentration. Increasing the histone concentration did not modify the inhibition. Although these studies suggest a novel cellular control mechanism, the high thyroid hormone concentrations required and the lack of concordance between inhibitory effects and biologic activity of the analogs tested precludes assumption of physiologic relevance.
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PMID:Inhibition of thyroidal cyclic AMP-dependent protein kinase by thyroid hormone. 21 93

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

Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Ialpha and Ibeta) on DEAE-Sephadex A25. Fractions I, Ialpha, Ibeta comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the folling characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [3H]AMP showed similar mobilities for Ialpha, Ibeta or II (Rf 0.37) whereas fraction III displayed a much greater mobility (RF 0.73); Scatchard plots were linear for fractions Ialpha, II and III with an apparent Kd in the same range (2 to 5 nM) whereas fraction Ibeta generated a biphasic plot with Kd 0.4 nM and 20 nM; cyclic [3H] AMP added to fraction I, Ialpha or Ibeta generated a cyclic [3H] AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.
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PMID:Cyclic AMP-binding proteins and protamine kinases in porcine thyroid cytosol. 21 21

The human erythrocyte contains two types of cyclic AMP-dependent protein kinase. The membrane-associated protein kinase holoenzyme can be released intact by hypotonic treatment at alkaline pH. Chromatography on DEAE-cellulose and molecular weight determinations demonstrate that this enzyme is a type I cyclic AMP-dependent protein kinase, while the predominant cyclic AMP-dependent protein kinase found in the cytoplasm is type II. The catalytic subunits of the two types of kinase found in the erythrocyte are identical, but the regulatory subunits, which distinguish the two types of kinase, determine their localization within the cell. It is proposed that the regulatory subunit of the type I enzyme, in addition to binding the catalytic subunit, interacts specifically with one or more membrane proteins and that this interaction may serve to position the kinase in preferential proximity to protein substrates.
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PMID:Compartmentalization of cyclic AMP-dependent protein kinases in human erythrocytes. 21 2

1. DEAE-cellulose chromatography of mouse brain cytosol indicated the presence of only the type II isoenzyme of cyclic AMP-dependent protein kinase. Mouse heart cytosol contained approximately equal amounts of the type I and type II isoenzymes. 2. Both brain and heart type II isoenzymes reassociated after a transient exposure to cyclic AMP, but the heart type I isoenzyme remained dissociated. 3. Elution of brain cytosol continuously exposed to cyclic AMP resolved multiple peaks of protein kinase and cyclic AMP-binding activities. A single peak of kinase and multiple peaks of cyclic AMP-binding activities were found under the same conditions with heart cytosol. Various control experiments suggested that the heterogeneity within the brain type II isoenzymic class had not been caused by proteolysis. 4. Kinetic experiments with unfractionated brain cytosol showed that the binding of cyclic AMP, the dissociation of cyclic AMP from protein and the rate of heat denaturation of the cyclic AMP-binding activity gave results consistent with the presence of multiple binding species. 5. It concluded that the type II isoenzymic peak obtained by DEAE-cellulose chromatography of mouse brain cytosol represents a class of enzymes containing multiple regulatory and catalytic subunits. The two heart cytosol isoenzymes contain a common catalytic subunit. The degree of protein kinase 'microheterogeneity", defined as the presence of multiple regulatory and/or catalytic subunits within a single isoenzymic class, appears to be tissue-specific.
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PMID:Microheterogeneity of adenosine cyclic monophosphate-dependent protein kinases from mouse brain and heart. 21 38

Cyclic nucleotide dependent protein kinase has been extracted wiht Tris or Lubrol PX from purified rod outer segments (ROS) of bovine retina. The activity of the enzyme is unaffected by light but is stimulated by either cyclic guanosine 3',5'-monophosphate (cGMP) or cyclic adenosine 3',5'-monophosphate (cAMP). Most of the solubilized enzyme elutes from DEAE-cellulose with about 0.18 M NaCl (type II protein kinase). An endogenous 30,000 molecular weight protein of the soluble fraction of ROS as well as exogenous histone are phosphorylated by the protein kinase in a cyclic nucleotide dependent manner. The Tris-extracted enzyme can be reassociated in the presence of Mg2+ with ROS membranes that are depleted of protein kinase activity. The reassociated protein kinase is insensitive to exogenous cyclic nucleotides, and it catalyzes the phosphorylation of the membrane protein, bleached rhodopsin. While the soluble and membrane-associated protein kinases may be interchangeable, they appear to be modulated by different biological signals; soluble protein kinase activity is increased by cyclic nucleotides whereas membrane-bound activity is enhanced when rhodopsin is bleached by light.
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PMID:Cyclic nucleotide dependent protein kinase and the phosphorylation of endogenous proteins of retinal rod outer segments. 21 12

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

A protein antigenically related to the simian virus (SV 40) A gene product has been purified to near homogeneity from cells infected with the adenovirus-SV 40 hybrid virus Ad2(+)D2 and shown to contain ATPase (ATP phosphohydrolase, EC 3.6.1.3) and protein kinase (ATP:phosphotransferase, EC 2.7.1.37) activity. Both enzymatic activities copurify with the protein through six stages including one gel filtration column, two ion exchange columns, and a heparin affinity column. Analogous fractions from extracts of cells uninfected or infected with adenovirus 2 alone do not contain these enzymatic activities. The D2 hybrid protein resolves into two forms (I and II) during ion exchange chromatography. Form I, the major species (85%) of the D2 hybrid protein, elutes from DEAE-Sephadex in 0.37 M NaCl and is able to catalyze the hydrolysis of ATP to ADP + P(i) at a rate of 3 mumol/hr per mg. The remaining 10-15% of the D2 hybrid protein consists of form II which elutes from DEAE-Sephadex in 0.29 M NaCl and is able to hydrolyze ATP as well as to incorporate phosphorus from ATP into either the D2 hybrid protein itself or other protein acceptors such as phosvitin. Although both forms are able to bind DNA, the ATPase activity of form I cosediments with SV 40 DNA more efficiently than does the protein kinase activity of form II during glycerol gradient centrifugation. The ATPase activity of form I is efficiently inhibited by addition of anti-T gamma globulin to the reaction mixture whereas control gamma globulin has no effect. Similarly, the phosphorylation of the D2 hybrid protein by form II is inhibited by anti-T gamma globulin. By contrast, phosphorylation of phosvitin is specifically inhibited by antibody only when the immune complex is removed from the reaction mixture. Thus, it appears likely that one and possibly two enzymatic activities are carried out by the D2 hybrid protein. These findings are discussed in terms of mechanisms of SV 40 DNA replication and virally induced transformation.
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PMID:Enzymatic activities associated with a purified simian virus 40 T antigen-related protein. 21 12


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