Rat Liver Nuclear Protein Kinases

1975 ◽  
Vol 53 (3) ◽  
pp. 354-363 ◽  
Author(s):  
P. R. Desjardins ◽  
C. C. Liew ◽  
A. G. Gornall
Keyword(s):  
Ionic Strength ◽  
Cyclic Amp ◽  
Dehydrogenase Activity ◽  
Kinase Activity ◽  
Nuclear Material ◽  
Casein Kinase ◽  
Casein Kinases ◽  
Liver Nuclei ◽  
Histone Kinase ◽  
Low Ionic Strength

We have shown that nuclei isolated by two methods contain grossly different amounts of cyclic AMP-dependent histone kinase activity. Repeated washing of the isolated nuclei with a low ionic strength buffer removed the majority of the cyclic AMP-dependent histone kinase and cyclic AMP binding activity. Nuclear cyclic AMP-dependent histone kinase activity accounted for only 0.42% of the total cytoplasmic enzyme activity. Similarly, the lactate dehydrogenase activity associated with liver nuclei represented only 0.07% of the total cytoplasmic activity. The isolated liver nuclei contained only 0.27% of the total homogenate glutamate dehydrogenase activity and 1.7% of the total homogenate glucose-6-phosphatase activity. The cyclic AMP-dependent histone kinase behaves as a cytoplasmic rather than a nuclear enzyme. We have also shown that using crude extracts, one can achieve separation of the two nuclear casein kinases, NI and NII, on sucrose density gradients in the presence of 0.5 M NaCl. Nuclear casein kinases NI and NII had sedimentation coefficients of 3.0 and 5.3 S, respectively, in the presence of 0.5 M NaCl. Under conditions of low ionic strength, all of the casein kinase activity in the crude nuclear extract sedimented as one peak with a sedimentation coefficient of 7.3 S. The aggregation–disaggregation which occurred in the crude extract was reversible and was mainly due to the aggregative and disaggregative properties of casein kinase NII The two nuclear casein kinases have different affinities for chromatin. When nuclei were disrupted in a hypotonic solution and extracted with a buffer containing 0.14 M NaCl, casein kinase NII could be completely extracted from the viscous nuclear material. Although a significant amount of casein kinase NI was extracted by the buffer containing 0.14 M NaCl, re-extraction of the nuclear material with a buffer containing 0.5 M NaCl yielded substantial amounts of casein kinase NI, and a final extraction with a buffer containing 1.0 M NaCl yielded measurable amounts of casein kinase NI. No casein kinase NII activity could be detected in the 0.5 M and 1.0 M NaCl extracts.

scholarly journals Phosphorylation by casein kinase II affects the interaction of caldesmon with smooth muscle myosin and tropomyosin

1993 ◽  
Vol 290 (2) ◽  
pp. 437-442 ◽  
Author(s):  
N V Bogatcheva ◽  
A V Vorotnikov ◽  
K G Birukov ◽  
V P Shirinsky ◽  
N B Gusev
Keyword(s):  
Smooth Muscle ◽  
Ionic Strength ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Smooth Muscle Myosin ◽  
Myosin Phosphorylation ◽  
Chymotryptic Peptide ◽  
Molecular Masses ◽  
Low Ionic Strength ◽  
Muscle Myosin

Smooth muscle caldesmon was phosphorylated by casein kinase II, and the effects of phosphorylation on the interaction of caldesmon and its chymotryptic peptides with myosin and tropomyosin were investigated. The N-terminal chymotryptic peptide of caldesmon of molecular mass 27 kDa interacted with myosin. Phosphorylation of Ser-73 catalysed by casein kinase II resulted in a 2-fold decrease in the affinity of the native caldesmon (or its 27 kDa N-terminal peptide) for smooth muscle myosin. At low ionic strength, caldesmon and its N-terminal peptides of molecular masses 25 and 27 kDa were retarded on a column of immobilized tropomyosin. Phosphorylation of Ser-73 led to a 2-4-fold decrease in the affinity of caldesmon (or its N-terminal peptides) for tropomyosin. Thus phosphorylation of Ser-73 catalysed by casein kinase II affects the interaction of caldesmon with both smooth muscle myosin and tropomyosin.

scholarly journals Effects of vanadate on protein kinases in rat hepatocytes

1989 ◽  
Vol 262 (2) ◽  
pp. 563-567 ◽  
Author(s):  
C Villar-Palasi ◽  
J J Guinovart ◽  
A M Gómez-Foix ◽  
J E Rodriguez-Gil ◽  
F Bosch
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Activity Ratio ◽  
Glycogen Synthase ◽  
Rat Hepatocytes ◽  
Casein Kinase ◽  
Optimal Time ◽  
Dependent Protein Kinase ◽  
Dependent Protein

In rat hepatocytes, vanadate modifies neither the intracellular concentration of cyclic AMP nor the -cyclic AMP/+cyclic AMP activity ratio for cyclic AMP-dependent protein kinase. Vanadate can, however, counteract the increase in cyclic AMP and the increase in the -cyclic AMP/+cyclic AMP activity ratio of cyclic AMP-dependent protein kinase induced by glucagon. On the other hand, vanadate treatment of hepatocytes can produce a time- and concentration-dependent increase in cyclic AMP- and Ca2+-independent casein kinase activity. Maximal activation at the optimal time with 5 mM-vanadate was about 70% over control. A clear relationship was observed between the activation of casein kinase and the inactivation of glycogen synthase after vanadate treatment. These results suggest that casein kinase activity may be involved in vanadate actions in rat hepatocytes.

scholarly journals Protein kinase activity in commercially available crystalline yeast alcohol dehydrogenase

1973 ◽  
Vol 131 (2) ◽  
pp. 271-274 ◽  
Author(s):  
B. E. Kemp ◽  
M. Froscio ◽  
A. W. Murray
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Alcohol Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Density Centrifugation ◽  
Yeast Alcohol Dehydrogenase ◽  
Alcohol Dehydrogenase Activity ◽  
Calf Thymus

Commercially available crystalline yeast alcohol dehydrogenase contained protein kinase activity. Casein and phosvitin were readily phosphorylated, but whole calf thymus histone was not. The protein kinase activity was inhibited by KCl, was not stimulated by cyclic AMP and could be separated from the alcohol dehydrogenase activity by sucrose density centrifugation.

scholarly journals Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber

1975 ◽  
Vol 149 (2) ◽  
pp. 329-339 ◽  
Author(s):  
A R Ashton ◽  
G M Polya
Keyword(s):  
Ionic Strength ◽  
Cyclic Amp ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Higher Plants ◽  
Gel Filtration ◽  
Nucleotide Metabolism ◽  
Partial Purification ◽  
Higher Plant ◽  
Low Ionic Strength

1. Three phosphodiesterases that are capable of hydrolysing 3':5'-cyclic nucleotides were purified from potato tubers. 2. The phosphodiesterases were fractionated by (NH4)2SO4 precipitation and CM-cellulose chromatography. The phosphodiesterases were resolved from each other and further purified by gel filtration in high- and low-ionic-strength conditions. 3. All three enzymes lacked significant nucleotidase activity. 4. Enzymes I and II had mol. wts. 240,000 and 80,000 respectively, determined by gel filtration, whereas enzyme III showed anomalous behaviour on gel filtration, behaving as a high- or low-molecular-weight protein in high- or low-ionic-strength buffers respectively. 5. All enzymes hydrolysed 2':3'-cyclic nucleotides as well as 3':5'-cyclic nucleotides. The enzymes also had nucleotide pyrophosphatase activity, hydrolysing NAD+ and UDP-glucose to various extents. Enzymes I and II hydrolyse cyclic nucleotides at a greater rate than NAD+, whereas enzyme III hydrolyses NAD+ at a much greater rate than cyclic nucleotides. All three enzymes hydrolysed the artificial substrate bis-(p-nitro-phenyl) phosphate. 6. The enzymes do not require the addition of bivalent cations for activity. 7. Both enzymes I and II have optimum activity at pH6 with 3':5'-cyclic AMP and bis-(p-nitrophenyl) phosphate as substrates. The products of 3':5'-cyclic AMP hydrolysis were 3'-AMP and 5'-AMP, the ratio of the two products being different for each enzyme and varying with pH. 8. Theophylline inhibits enzymes I and II slightly, but other methyl xanthines have little effect. Enzymes I and II were competitively inhibited by many nucleotides containing phosphomonoester and phosphodiester bonds, as well as by Pi. 9. The possible significance of these phosphodiesterases in cyclic nucleotide metabolism in higher plants is discussed.

scholarly journals Changes in histone phosphorylation and associated early metabolic events in pig lymphocyte cultures transformed by phytohaemagglutinin or 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate

1971 ◽  
Vol 124 (1) ◽  
pp. 241-248 ◽  
Author(s):  
M E. Cross ◽  
M G. Ord
Keyword(s):  
Cyclic Amp ◽  
Kinase Activity ◽  
Specific Radioactivity ◽  
Dibutyryl Cyclic Amp ◽  
Kinase Activation ◽  
Cyclic Monophosphate ◽  
Lymphocyte Cultures ◽  
Histone Kinase ◽  
Rna And Dna ◽  
Phosphate Groups

1. Pig lymphocytes were transformed by dibutyryl cyclic AMP (6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) at concentrations of 0.01–0.1μm. The pattern of incorporation of label from [5-3H]uridine and [6-3H]thymidine into RNA and DNA respectively was identical with that obtained with unpurified phytohaemagglutinin. 2. Chlorpromazine (0.1μm) prevented the stimulation of [5-3H]uridine incorporation into RNA by phytohaemagglutinin, but only slightly lowered the lymphocyte response to dibutyryl cyclic AMP. 3. An increase in the size and specific radioactivity of the intracellular Pi pool was found immediately after stimulation by both phytohaemagglutinin and dibutyryl cyclic AMP. This was followed after some 30min by a rise in the specific radioactivity and concentration of ATP. 4. There was an immediate increase in the specific radioactivity of phosphate groups of histones; by about 45min after stimulation only the histones remaining after extraction of histone fraction F1 continued to incorporate 32P from [32P]Pi. 5. Histone kinase activity increased in the first 30min after stimulation; subsequently histone F1 kinase activity decreased, but activity with the other histones as substrate continued to increase for a further 30min. Kinase activation was effected by cyclic AMP (adenosine 3′:5′-cyclic monophosphate). 6. Histone phosphatase activity behaved similarly to that of the kinase.

scholarly journals Role of histone kinases as mediators of corticotropin-induced steroidogenesis*

1976 ◽  
Vol 160 (1) ◽  
pp. 1-9 ◽  
Author(s):  
W R Moyle ◽  
G J MacDonald ◽  
J E Garfink
Keyword(s):  
Cyclic Amp ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Dibutyryl Cyclic Amp ◽  
Cyclic Amp Accumulation ◽  
Calf Thymus ◽  
High Doses ◽  
Histone Kinase ◽  
Very High

In an attempt to determine the role of protein (histone) kinases as mediators of corticotropin-induced corticosterone formation, the ability of homogenates, prepared from adrenals treated with various doses of corticotropin to catalyse the phosphorylation of calf thymus histones was measured. Although corticotropin promoted an increase in histone kinase activity, much more of the hormone was required to induce this response than to stimulate steroidogenesis maximally. In addition, a derivative, nitrophenylsulphenyl-corticotropin, which inhibits the stimulatory effect of corticotropin on cyclic AMP accumulation, stimulated corticosterone synthesis without altering histone kinase activity. Very high doses of nitrophenylsulphenyl-corticotropin were capable of stimulating histone kinase activity. In contrast, when dibutyryl cyclic AMP was used to stimulate steroidogenesis under the same conditions, any dose of the nucleotide which increased adrenal corticosteroid content also increased histone kinase activity. Assuming that histones serve as useful substrates for measurement of total adrenal protein kinase activity, the role of protein kinases as mediators of steroidogenesis is not supported by these studies.

scholarly journals Differing accessibility in chromatin of the antigenic sites of regions 1-58 and 63-125 of histone H2B.

1981 ◽  
Vol 91 (1) ◽  
pp. 135-141 ◽  
Author(s):  
D di Padua Mathieu ◽  
C V Mura ◽  
L L Frado ◽  
C L Woodcock ◽  
B D Stollar
Keyword(s):  
Ionic Strength ◽  
Staining Intensity ◽  
Histone H2b ◽  
Antigenic Sites ◽  
Liver Nuclei ◽  
Low Ionic Strength

Experiments with antibodies induced by separated fragments 1-58 and 63-125 of H2B histone indicated that the 1-58 portion of the molecule is much more accessible in chromatin than is the 63-125 region. In immunoabsorption and immunoelectron microscopic assays with bovine and chicken chromatins, anti-1-58 antibodies reacted with sheared or unsheared chromatin both at low ionic strength (1 mM Tris-HCl) and in 0.14 M NaCl. Anti-63-125 antibodies were bound only weakly by chromatin at low ionic strength and not at all in 0.14 M NaCl. Antibodies to whole H2B showed intermediate reactivity with chromatin in both assays. In tests of immunofluorescence with unfixed calf liver nuclei in suspension, anti-1-58 caused nucleolar as well as nucleoplasmic fluorescence, whereas anti-63-125 did not lead to detectable fluorescence; anti-H2B showed intermediate staining intensity. In control experiments, anti-H1 antibody was bound by chromatin at low ionic strength but not in 0.14 M NaCl; anti-H3 antibody was bound poorly under either condition.

scholarly journals Multiple protein kinases from human lymphocytes. Identification enzymes phosphorylating exogenous histon and casein

1975 ◽  
Vol 145 (2) ◽  
pp. 241-249 ◽  
Author(s):  
B E Kemp ◽  
M Froscio ◽  
A Rogers ◽  
A W Murray
Keyword(s):  
Molecular Weight ◽  
Cyclic Amp ◽  
Kinase Inhibitor ◽  
Muscle Protein ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Casein Kinase ◽  
Rabbit Muscle ◽  
Sephadex Column ◽  
Histone Kinase

1. Cell-free lysates of human peripheral blood lymphocytes contained two casein kinase activities and two histone kinase activities, which could be separated by chromatography on DEAE-Sephadex. 2. Neither of the casein kinase activities were stimulated by cyclic AMP. The major activity was eluted from DEAE-Sephadex between 0.4 and 0.45M-KCl, had a molecular weight of approx. 130,000 (sucrose density gradients) and was stimulated by KCl (maximum 150mM). It also formed higher-molecular-weight aggregates when centrifuged in sucrose gradients containing 150mM-KCl. The minor activity was not retained by DEAE-Sephadex, had a molecular weight of approx. 50,000 and was not stimulated by KCl. 3. The major histone kinase activity was stimulated by cyclic AMP and was eluted from the DEAE-Sephadex column between 0.05 and 0.2M-KCl. The other activity was not stimulated by cyclic AMP and was insensitive to the rabbit muscle protein kinase inhibitor. 4. Evidence was obtained suggesting that the lymphocyte casein kinases were located primarily in the nuclei.

scholarly journals Experimental conditions affecting ribonucleic acid polymerase in isolated rat liver nuclei. Effect of nucleoside triphosphate concentration, temperature, ammonium sulphate and heparin

1969 ◽  
Vol 112 (5) ◽  
pp. 721-727 ◽  
Author(s):  
F. Novello ◽  
F. Stirpe
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
High Ionic Strength ◽  
Isolated Rat Liver ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Low Ionic Strength ◽  
Isolated Rat

1. The conditions affecting the activity of RNA polymerase in isolated rat liver nuclei were studied with Mg2+ or Mn2+ as activating ions. 2. The enzyme assayed with Mg2+ and at low ionic strength is saturated by a lower concentration of nucleotide substrates than if assayed with Mn2+ at low ionic strength or with either ion at high ionic strength. 3. At low and at high ionic strength the incorporation of AMP is affected in a similar way by variations in the temperature of incubation. Preincubation at 37° impairs the AMP incorporation. 4. Heparin stimulates the RNA polymerase activity in the presence of Mn2+. 5. Both ammonium sulphate and heparin ‘restart’ the reaction if added after 15min., the effect being more marked with ammonium sulphate than with heparin, and also more marked in the presence of Mn2+ than of Mg2+. 6. α-Amanitin abolishes the effect of ammonium sulphate and of heparin.

The ribosomal proteins phosphorylated in vitro by protein kinase activities from Krebs II ascites cells

1983 ◽  
Vol 3 (7) ◽  
pp. 621-629 ◽  
Author(s):  
Michael J. McGarvey ◽  
David P. Leader
Keyword(s):  
Protein Kinase ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Kinase Activity ◽  
Casein Kinase ◽  
Protein Kinase Activity ◽  
Similar Reaction ◽  
Histone Kinase

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.

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