scholarly journals Adenosine 3':5'-cyclic monophosphate-dependence of protein kinase isoenzymes from mouse liver

1976 ◽  
Vol 157 (1) ◽  
pp. 117-126 ◽  
Author(s):  
P M Ueland ◽  
S O Døskeland
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Mouse Liver ◽  
Deae Cellulose ◽  
Maximal Activity ◽  
Close Correlation ◽  
Histone Phosphorylation ◽  
Heat Stable ◽  
Dependent Protein ◽  
High Concentrations

Conditions influencing the cyclic AMP-dependence of protein kinase (ATP-protein phosphotransferase, EC 2.7.1.37) during the phosphorylation of histone were studied. Protein kinase from mouse liver cytosol and the two isoenzymes [PK (protein kinase) I and PK II] isolated from the cytosol by DEAE-cellulose chromatography were tested. A relation between concentration of enzyme and cyclic AMP-dependence was observed for both isoenzymes. Moderate dilution of isoenzyme PK II decreased the stimulation of the enzyme by cyclic AMP. Isoenzyme PK I could be diluted 200 times more than isoenzyme PK II before the same decrease in cyclic AMP-dependence appeared. Long-term incubation with high concentrations of histone increased the activity in the absence of cyclic AMP relative to the activity in the presence of the nucleotide. This was more pronounced for isoenzyme PK II than for isoenzyme PK I. The cyclic AMP concentration needed to give half-maximal binding of the nucleotide was the same as the cyclic AMP concentration (Ka) at which the protein kinase had 50% of its maximal activity. The close correlation between binding and activation is also found in the presence of KCl, which increased the apparent activation constant (Ka) for cyclic AMP. With increasing [KCl], a progressively higher proportion of the histone phosphorylation observed in cytosol was due to cyclic AMP-independent (casein) kinases, leading to an overestimation of the degree of activation of the cyclic AMP-dependent protein kinases present. The relative contributions of cyclic AMP-dependent and -independent kinases to histone phosphorylation at different ionic strengths was determined by use of heat-stable inhibitor and phospho-cellulose chromatography.

scholarly journals Microheterogeneity of adenosine cyclic monophosphate-dependent protein kinases from mouse brain and heart

1978 ◽  
Vol 175 (2) ◽  
pp. 367-375 ◽  
Author(s):  
A M Malkinson ◽  
A J Gharrett ◽  
L Hogy
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Mouse Brain ◽  
Deae Cellulose ◽  
Type I ◽  
Type Ii ◽  
Catalytic Subunits ◽  
Multiple Peaks ◽  
Dependent Protein ◽  
Brain Cytosol

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.

scholarly journals Properties of neurofilament protein kinase

1986 ◽  
Vol 235 (1) ◽  
pp. 283-289 ◽  
Author(s):  
D Toru-Delbauffe ◽  
M Pierre ◽  
J Osty ◽  
F Chantoux ◽  
J Francon
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Specific Activity ◽  
Maximal Activity ◽  
Brain Cell ◽  
Neurofilament Protein ◽  
Dependent Protein ◽  
Protein Components ◽  
Cytoskeletal Elements

Neurofilament (NF) protein kinase, partially purified from NF preparations [Toru-Delbauffe & Pierre (1983) FEBS Lett. 162, 230-234], was found to be distinct from both the casein kinase present in NFs and the cyclic AMP-dependent protein kinase which is able to phosphorylate NFs. NF-kinase phosphorylated the three NF protein components. The amount of phosphate incorporated per molecule was higher for NF 200 than for NF 145 and NF 68. Other proteins present in the NF preparations were also used as NF-kinase substrates. Two of them might correspond to the myelin basic proteins with Mr values of 18,000 and 21,000. Four other substrates in the NF preparation were not identified (respective Mr values 53,000, 55,000, 65,000 and greater than 300,000). NF kinase also phosphorylated two additional brain-cell cytoskeletal elements: GFAp and vimentin. Casein, histones and phosvitin, currently used as substrates for protein kinase assays, were very poor phosphate acceptors. Half-maximal NF-kinase activity was obtained at an NF protein concentration of about 0.25 mg/ml in heated, salt-washed, NF preparations. The specific activity was about 5 pmol of 32P incorporated/min per microgram of NF kinase preparation protein. ATP was a phospho-group donor (Km 8 × 10(-5) M), but GTP was not. NF-kinase activity remained stable at 65 degrees C for more than 1 h. The enzyme was not degraded by storage at −20 degrees C for several months in a buffer containing 50% (w/v) sucrose. Maximal activity was obtained with 5 mM-Mg2+ (Mg2+ could be replaced by Co2+); Zn2+ and Cu2+ inhibited the reaction. NF-kinase was not dependent on cyclic AMP, cyclic GMP, Ca2+ or Ca2+ plus dioleoylglycerol and phosphatidylserine.

Adenosine-3′,5′-Monophosphate-Dependent Protein Kinase From Bovine Anterior Pituitary Gland. III. Structural Specificity of the ATP Site of the Catalytic Subunit

1974 ◽  
Vol 52 (2) ◽  
pp. 137-141 ◽  
Author(s):  
Simon Lemaire ◽  
Fernand Labrie ◽  
Marie Gauthier
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Catalytic Subunit ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Stringent Requirement ◽  
Dependent Protein ◽  
Atp Analogues ◽  
High Concentrations ◽  
Derivatives Of

The effect of analogues and derivatives of adenosine 3′,5′-monophosphate (cyclic AMP) and of ATP on the incorporation of 32P from [γ-32P]ATP into histones has been measured using the purified catalytic subunit of adenohypophyseal protein kinase. Gamma-labeled CTP, GTP, and UTP cannot substitute for [γ-32P]ATP but they slightly inhibit the phosphorylation by [γ-32P]-ATP when present as unlabeled compounds. A stringent requirement of the adenine nucleus is observed for the ability to compete at the ATP site, inhibitions of 42, 39, 32, and 63% being observed respectively with adenine, adenosine, 5′AMP, and ADP, while the corresponding purine or pyrimidine derivatives have no effect when present at a 13-fold molar excess relative to [γ-32P]ATP. The N6-benzoyl and N6-butyryl derivatives of cyclic AMP are inactive whereas the 8-substituted derivatives are generally as active as cyclic AMP itself, except for the 8-amino- and 8-hydroxy-derivatives, which exhibit a lower degree of competition. All cyclic AMP and ATP analogues and derivatives that inhibit histone phosphorylation by [γ-32P]ATP act as competitive inhibitors. Such competition at the ATP site of the catalytic subunit of protein kinase probably accounts for the progressive inhibition of cyclic-AMP-dependent protein kinase activity measured at high concentrations (above 10−5 M) of the cyclic nucleotide.

scholarly journals Characterization of the protein kinase activities of human platelet supernatant and particulate fractions

1984 ◽  
Vol 218 (2) ◽  
pp. 285-294 ◽  
Author(s):  
S E Salama ◽  
R J Haslam
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Soluble Fraction ◽  
Deae Cellulose ◽  
Type I ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Regulatory Subunits ◽  
Dependent Protein ◽  
Triton X

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.

scholarly journals Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase

1993 ◽  
Vol 13 (8) ◽  
pp. 4477-4484
Author(s):  
E Kupperman ◽  
W Wen ◽  
J L Meinkoth
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Dna Synthesis ◽  
Kinase Inhibitor ◽  
Dependent Protein Kinase ◽  
Thyroid Cells ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Dependent Protein ◽  
Rat Thyroid

Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.

scholarly journals Guanosine cyclic monophosphate-dependent protein kinase from foetal calf heart. Purification, general properties and catalytic subunit

1977 ◽  
Vol 161 (2) ◽  
pp. 213-221 ◽  
Author(s):  
M Shoji ◽  
J G Patrick ◽  
C W Davis ◽  
J F Kuo
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Deae Cellulose ◽  
Catalytic Subunit ◽  
Subunit Structure ◽  
Dependent Protein Kinase ◽  
Ph Optimum ◽  
General Properties ◽  
Dependent Protein

Cyclic GMP-dependent protein kinase was purified from foetal calf hearts, and its general properties and subunit structure were studied. The enzyme was purified over 900-fold from the heart extract by pH 5.3-isoelectric precipitation, DEAE-cellulose chromatography, Sephadex G-200 filtration and hydroxyapatite treatment. The purified myocardial enzyme, free from cyclic AMP-dependent protein kinase contamination, exhibited an absolute requirement of stimulatory modulator (or crude modulator containing the stimulatory modulator component) for its cyclic GMP-stimulated activity. Inhibitory modulator (protein inhibitor) of cyclic AMP-dependent protein kinase could not stimulate nor inhibit the cyclic GMP target enzyme. The enzyme had Ka values of 0.013, 0.033 and 3.0 micronM for 8-bromo cyclic GMP, cyclic GMP and cyclic AMP respectively. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity, with optimal concentrations of about 30 and 0.5 mM respectively. The pH optimum for the enzyme activity ranged from 6 to 9. Histones were generally effective substrate proteins. The enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent class of protein kinase. The holoenzyme (apparent mol.wt. 150 000) of the myocardial cyclic GMP-dependent protein kinase was dissociated into a cyclic GMP-independent catalytic subunit (apparent mol.wt. 60 000) by cyclic GMP and histone. The catalytic subunit required the stimulatory modulator for its activity, as in the case of the holoenzyme in the presence of cyclic GMP.

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