Purification and Properties of Rat Liver Nuclear Protein Kinases

1972 ◽  
Vol 50 (12) ◽  
pp. 1249-1259 ◽  
Author(s):  
P. R. Desjardins ◽  
P. F. Lue ◽  
C. C. Liew ◽  
A. G. Gornall
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Rat Liver ◽  
Nuclear Protein ◽  
Kinase Inhibitor ◽  
Divalent Cations ◽  
Ph Optimum ◽  
Heat Stable ◽  
Low Concentrations

Two protein kinases, designated NI and NII, have been isolated from rat liver nuclei. These enzymes have a similar pH optimum and phosphorylate phosvitin and casein more readily than histone. Both enzymes require magnesium for activity. In the absence of Mg2+, other divalent cations such as Ca2+, Co2+, and Mn2+ can substitute partially for Mg2+ when the reaction is catalyzed by NI. With NII, only Co2+ showed any activity in the absence of Mg2+. Magnesium decreased the apparent Km for ATP of protein kinase NI without changing the Vmax of the reaction, and decreased the apparent Km's for both ATP and casein, while increasing the Vmax of the reaction threefold with protein kinase NII. Both enzymes are stimulated about twofold by low concentrations (0.1–0.3 M) of NaCl, KCl, and sodium acetate, whereas higher concentrations (> 0.5 M) inhibit their activities. Both enzymes are inhibited by low concentrations of NaF (0.02 M) and (NH4)2SO4 (0.1 M). NI and NII were found to have sedimentation coefficients of 3.6 S and 10.8 S, respectively. The nuclear protein kinases are not activated by cyclic AMP or cyclic GMP, and are not inhibited by the heat-stable cyclic AMP-dependent protein kinase inhibitor.

scholarly journals Purification and properties of a nuclear protein kinase from rat mammary gland

1977 ◽  
Vol 165 (3) ◽  
pp. 469-477 ◽  
Author(s):  
Gopal C. Majumder
Keyword(s):  
Mammary Gland ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Nuclear Protein ◽  
Kinase Inhibitor ◽  
Sedimentation Coefficient ◽  
Molecular Size ◽  
Regulatory Subunit ◽  
Heat Stable ◽  
Exclusion Chromatography

A nuclear protein kinase that shows a high degree of substrate specificity for the phosphorylation of the acidic proteins casein, phosvitin and non-histone chromatin proteins, rather than the basic proteins histones and protamine, was partially purified from lactatingrat mammary gland. The enzyme is associated with the acidic protein fraction of chromatin. Nuclear kinase requires Co2+ for activity, and other bivalent cations such as Mg2+ and Mn2+ can substitute partially for Co2+. The kinase is further activates (2–3-fold) by various salts, their concentration for maximum stimulation being: NaCl, 150mm; KCl, 200mm; sodium acetate, 300mm. The sedimentation coefficient of the nuclear kinase is 8.9S and its mol.wt. is approx. 300000 by gel-exclusion chromatography. The enzyme is not activated by cyclic AMP or cyclic GMP and is inhibited neither by the regulatory subunit of mammary cyclic AMP-dependent protein kinase nor by the heat-stable protein kinase inhibitor from ox heart. Analysis of 32P-labelled protein products reveals that the kinase transfers the terminal phosphate of ATP to serine and threonine residues of proteins. The enzyme, however, has specificity for the phosphorylation of threonine in casein and serine in phosvitin. Molecular size and enzymic characteristics of the nuclear protein kinase are clearly different from those of the cytosol enzyme previously characterized.

Regulation of protein kinase by vasopressin in renal medulla in situ

1977 ◽  
Vol 232 (1) ◽  
pp. F50-F57
Author(s):  
T. P. Dousa ◽  
L. D. Barnes
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Renal Medulla ◽  
Protein Kinase Activity ◽  
Kinase Activation ◽  
Heat Stable ◽  
Protein Kinase Activation ◽  
Heat Stable Protein

Results of this study demonstrate that vasopressin activates protein kinase in intact renal medullary cells as detected by measurement of the (-cyclic AMP/+cyclic AMP) protein kinase activity ratios in freshly prepared tissue extracts (40,000 X g supernates) from bovine renal medullary slices. The activation of protein kinase was specific for vasopressin since parathyroid hormone, histamine, angiotensin II, or the inactive analog of vasopressin did not activate protein kinase. There was a direct correlation between the extent of protein kinase activation and the elevation in tissue levels of cyclic AMP elicited by increasing doses of vasopressin or with an increase in incubation time. The elevation of tissue cyclic AMP level and maximum activation of protein kinase reached maximum level at a vasopressin concentration of about 2 X 10(-9) M. Incubation of slices with vasopressin caused a dose-dependent decrease in the cyclic AMP-dependent protein kinase activity in the 40,000 X g supernate of homogenate from the renal medullary slices. This effect of vasopressin was specific for protein kinase since activity of lactate dehydrogenase or a specific [3H]colchicine-binding activity was not affected, and the decrease in the protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase activity extracted from 40,000 X g pellets of homogenate prepared from slices exposed to vasopressin. Results thus provide evidence that cyclic AMP-mediated protein kinase activation in the intact cells is an integral part of cellular response of the mammalian renal medulla to vasopressin.

scholarly journals Antiserum against the catalytic subunit of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase. Reactivity towards various protein kinases

1980 ◽  
Vol 192 (1) ◽  
pp. 223-230 ◽  
Author(s):  
G Schwoch ◽  
A Hamann ◽  
H Hilz
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Rat Liver ◽  
Catalytic Subunit ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Subunit C ◽  
Bovine Heart ◽  
Dependent Protein

An antiserum against the catalytic subunit C of cyclic AMP-dependent protein kinase, isolated from bovine heart type II protein kinase, was produced in rabbits. Reaction of the catalytic subunit with antiserum and separation of the immunoglobulin G fraction by Protein A-Sepharose quantitatively removed the enzyme from solutions. Comparative immunotitration of protein kinases showed that the amount of antiserum required to eliminate 50% of the enzymic activity was identical for pure catalytic subunit, and for holoenzymes type I and type II. The reactivity of the holoenzymes with the antiserum was identical in the absence or the presence of dissociating concentrations of cyclic AMP. Most of the holoenzyme (type II) remains intact when bound to the antibodies as shown by quantification of the regulatory subunit in the supernatant of the immunoprecipitate. Titration with the antibodies also revealed the presence of a cyclic AMP-independent histone kinase in bovine heart protein kinase I preparations obtained by DEAE-cellulose chromatography. Cyclic AMP-dependent protein kinase purified from the particulate fraction of bovine heart reacted with the antiserum to the same degree as the soluble enzyme, whereas two cyclic AMP-independent kinases separated from the particle fraction neither reacted with the antiserum nor influenced the reaction of the antibodies with the cyclic AMP-dependent protein kinase. Immunotitration of the protein kinase catalytic subunit C from rat liver revealed that the antibodies had rather similar reactivities towards the rat liver and the bovine heart enzyme. This points to a relatively high degree of homology of the catalytic subunit in mammalian tissues and species. Broad applicability of the antiserum to problems related to cyclic AMP-dependent protein kinases is thus indicated.

Structural origins of AGC protein kinase inhibitor selectivities: PKA as a drug discovery tool

2012 ◽  
Vol 393 (10) ◽  
pp. 1121-1129 ◽  
Author(s):  
Espen Åberg ◽  
Bjarte Lund ◽  
Alexander Pflug ◽  
Osman A.B.S.M. Gani ◽  
Ulli Rothweiler ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitor ◽  
Constitutive Activity ◽  
Inhibitor Selectivity ◽  
Kinase A

Abstract The era of structure-based protein kinase inhibitor design began in the early 1990s with the determination of crystal structures of protein kinase A (PKA, or cyclic AMP-dependent kinase). Although many other protein kinases have since been extensively characterized, PKA remains a prototype for studies of protein kinase active conformations. It serves well as a model for the structural properties of AGC subfamily protein kinases, clarifying inhibitor selectivity profiles. Its reliable expression, constitutive activity, simple domain structure, and reproducible crystallizability have also made it a useful surrogate for the discovery of inhibitors of both established and emerging AGC kinase targets.

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.

Protein kinase activities in rat liver nuclei: Effects of age and partial hepatectomy

1986 ◽  
Vol 6 (6) ◽  
pp. 565-571
Author(s):  
Debbie V. E. Cumming ◽  
Margery G. Ord ◽  
Lloyd A. Stocken
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Partial Hepatectomy ◽  
Rat Liver ◽  
Rat Liver Nuclei ◽  
Dependent Protein ◽  
Adrenergic Blockers ◽  
Liver Nuclei ◽  
Endogenous Proteins

Selective substrates and inhibitors have been used to measure kinases phosphorylating endogenous proteins in rat liver nuclei during growth and regeneration after partial hepatectomy. Peaks in activity were found at 5, 22, and 29 hours after partial hepatectomy. Administration of α1 and β adrenergic blockers suggested that the Be2+ sensitive and cyclic AMP-dependent protein kinases were interdependently regulated by Ca2+ and cyclic AMP.

scholarly journals Nuclear protein kinase activity in glucagon-stimulated perfused rat livers

1974 ◽  
Vol 143 (2) ◽  
pp. 469-471 ◽  
Author(s):  
Warren K. Palmer ◽  
Monique Castagna ◽  
Donal A. Walsh
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Nuclear Protein ◽  
Regulatory Subunit ◽  
Protein Kinase Activity ◽  
Heat Stable ◽  
Rat Livers

Nuclei isolated from glucagon-stimulated perfused rat livers contained 2–3 times as much protein kinase activity as did nuclei from control animals. In the presence of either the heat-stable inhibitor or the protein kinase regulatory subunit the elevated cyclic AMP-independent enzyme activity from stimulated nuclei was inhibited to an activity equivalent to that found in controls.

scholarly journals A cyclic nucleotide-independent protein kinase in Leishmania donovani

1986 ◽  
Vol 240 (3) ◽  
pp. 641-649 ◽  
Author(s):  
S Das ◽  
A K Saha ◽  
N K Mukhopadhyay ◽  
R H Glew
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
High Speed ◽  
Leishmania Donovani ◽  
Kinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dynamic State ◽  
Supernatant Fraction ◽  
Protein Kinase Activity ◽  
Ph Optimum

Leishmania donovani promastigotes labelled for 2 h with 32Pi incorporated radioactivity into at least 21 different proteins, as determined by SDS/polyacrylamide-gel electrophoresis. Pulse-chase studies with 32Pi demonstrated that the labelled proteins were in a dynamic state: some radiolabelled proteins rapidly disappeared and others appeared after the chase. The possibility of an ectokinase on the parasite was examined; incubation of intact parasites for 10 min at 25 degrees C in an osmotically buffered medium containing [gamma-32P]ATP, but not [alpha-32P]ATP, resulted in the labelling of 10 different protozoal proteins, presumably localized to the surface of the organism's plasma membrane. Intact promastigotes also catalysed the transfer of 32P from [gamma-32P]ATP to histones. The histone-dependent kinase was solubilized by repeated freezing and thawing, and sonication, and purified 118-fold by chromatographing the high-speed (200,000 g, 1 h) supernatant fraction on QAE-Sephadex, Sephadex G-150 and hydroxyapatite columns. The kinase eluted as a single activity peak from all three columns. The partially purified histone-dependent kinase had the following properties: pH optimum, 7.0; optimum temperature, 37 degrees C; Km for mixed calf thymus histone, 0.15 mM; Km for ATP, 0.8 mM; preferred fractionated histone acceptors, H2b greater than H4 greater than H2a greater than H3 (H1 does not serve as an acceptor); optimum activity required 10-20 mM-Mg2+; inhibited 50-80% by 0.01 mM- and 1 mM-Ca2+; activity was not stimulated by calmodulin, cyclic AMP (1 mM) or cyclic GMP (1 mM) nor inhibited by a cyclic AMP-dependent protein kinase inhibitor (50 micrograms/assay); apparent Mr 75,000, as determined by Sephadex G-150 gel filtration chromatography; phosphorylated exclusively serine residues. Protein kinase activity was low in the early exponential phase of the growth curve and increased 6-fold upon entry into the stationary phase.

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