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.

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.

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.

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 Modulation of nuclear cyclic AMP-dependent protein kinase in dibutyryl cyclic AMP-treated rat H4IIE hepatoma cells

1989 ◽  
Vol 260 (3) ◽  
pp. 673-682 ◽  
Author(s):  
S P Squinto ◽  
R A Jungmann
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Nuclear Protein ◽  
Fold Increase ◽  
Hepatoma Cells ◽  
Regulatory Subunit ◽  
Dibutyryl Cyclic Amp ◽  
Dependent Protein Kinase ◽  
C Subunit ◽  
Dependent Protein

Biochemical and immunochemical studies were undertaken to quantify the effects of cyclic AMP on cyclic AMP-dependent protein kinase subunit levels in nuclei of H4IIE hepatoma cells. Dibutyryl cyclic AMP (10 microM) caused a significant biphasic (10 and 120 min after stimulation) increase in total nuclear protein kinase activity. The increase observed 10 min after dibutyryl cyclic AMP stimulation was primarily due to an approx. 3-fold increase of catalytic (C) subunit activity, whereas the change observed 120 min after stimulation consisted of an increase in both C subunit and cyclic AMP-independent protein kinase activities. Analysis of nuclear protein extracts by photoaffinity labelling with 8-azido cyclic [32P]AMP identified only the type II regulatory subunit (RII), but not the type I regulatory subunit (RI). Analysis of nuclear RII variants by two-dimensional gel electrophoresis demonstrated that dibutyryl cyclic AMP caused the appearance of two RII variant forms which were not present in the nuclei of unstimulated cells. Using affinity-purified polyclonal antibodies and immunoblotting procedures, we identified an approx. 2-fold increase in the RII and C subunits in nuclear extracts of dibutyryl cyclic AMP-treated hepatoma cells. Finally, the RI, RII and C subunits were quantified by an e.l.i.s.a. which indicated that dibutyryl cyclic AMP increased nuclear RII and C subunits levels biphasically, reaching peak values 10 and 120 min after the initial stimulation. Nuclear RI subunit levels were not affected. These results provide qualitative as well as quantitative evidence for a modulation by cyclic AMP of the nuclear RII and C subunit levels in rat H4IIE hepatoma cells, and indicate a relatively rapid but temporarily limited dibutyryl cyclic AMP-induced translocation of the RII and C subunits to nuclear sites.

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 Regulation of acetyl-CoA carboxylase in rat mammary gland. Effects of incubation with Ca2+, Mg2+ and ATP on enzyme activity in tissue extracts

1981 ◽  
Vol 200 (3) ◽  
pp. 639-644 ◽  
Author(s):  
E M McNeillie ◽  
R A Clegg ◽  
V A Zammit
Keyword(s):  
Mammary Gland ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Inhibitor ◽  
Enzyme Activation ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Rat Mammary Gland ◽  
Subsequent Activation

1. The effect of preincubation of extracts of lactating rat mammary gland with ATP, Mg2+ and micromolar concentrations of Ca2+ on the activity of acetyl-CoA carboxylase was studied. 2. Both Mg2+ and Ca2+ activated the enzyme. Activation with Mg2+ (5 mM) was larger than that with Ca2+ (calculated free Ca2+ concentration = 20-50 microM), but the activity decreased after reaching a peak. The activation obtained with Ca2+ was stable for up to 180 min. 3. Incubation with Ca2+ and Mg2+ together resulted in an activation that was slightly higher than that with Mg2+ only and was stable (compare the results for Ca2+ alone). 4. Preincubation in the absence of Mg2+, but not in the absence of Ca2+, resulted in the impairment of subsequent activation with either Mg2+ (when preincubation was with Ca2+ alone) or Mg2+ plus Ca2+. 5. KF (50 mM) prevented the activation of acetyl-CoA carboxylase by Ca2+ and Mg2+. 6. MgATP2- reversed (Mg2+ + Ca2+)-mediated activation and decreased the activity of acetyl-CoA carboxylase to about 10% of initial activity. Inhibition by ATP was unaffected by addition of cyclic AMP or cyclic AMP-dependent protein kinase inhibitor. 7. 32P was incorporated into acetyl-CoA carboxylase when incubations were carried out in the presence of [gamma-32P]ATP. Subsequent removal of ATP from the incubation medium resulted in rapid loss of 32P from acetyl-CoA carboxylase. 8. It is suggested that extracts of rat mammary gland contain endogenous protein kinase and phosphatase activities that modulate acetyl-CoA carboxylase activity through reversible phosphorylation and dephosphorylation. The phosphatase activity is sensitive to both Mg2+ and micromolar concentrations of Ca2+, whereas the kinase does not appear to be cyclic AMP-dependent.

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.

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