scholarly journals Cyclic nucleotide-independent protein kinases bound to cytoplasmic and nuclear polyribosomes in non-infected and adenovirus-infected HeLa cells

1980 ◽  
Vol 189 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Mukti H. Sarma ◽  
Nando K. Chatterjee
Keyword(s):  
Protein Kinase ◽  
Hela Cells ◽  
Cyclic Nucleotide ◽  
Kinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Protein Kinase Activity ◽  
Cytoplasmic Proteins ◽  
Endogenous Substrates ◽  
Independent Protein

Cyclic nucleotide-independent protein kinase activity bound to cytoplasmic and nuclear polyribsomes from non-infected and adenovirus-infected HeLa cells was compared. The enzymes catalysed the incorporation of phosphate from γ-32P-labelled ATP or GTP into acid-precipitable material in the absence of exogenous substrates. Their activity was not affected by cyclic AMP or cyclic GMP and was not inhibited by a cyclic nucleotide-dependent protein kinase-inhibitor protein. The kinases are tightly bound to polyribosomes of either origin from infected and non-infected cells, since treatment with 0.5m-NaCl did not dissociate the activity. The enzymes and the enzyme-associated endogenous substrates of cytoplasmic polyribosomes are significantly different from those of the nucleus, and adenovirus infection of the cells did not alter the nature of the enzymes or the substrates at 18–20h after infection. Nuclear kinases catalysed 3–4-fold more phosphate incorporation than did the cytoplasmic kinases. They did not phosphorylate endogenous substrates in the cytoplasmic preparations, and vice versa, which suggests that such substrates for cytoplasmic and nuclear kinases are specific. Polyacrylamide-gel electrophoresis of the phosphorylated proteins revealed the presence of a higher number of endogenous substrates in the nuclear preparation. The nuclear kinases phosphorylated all histones from HeLa cells, but the cytoplasmic ones phosphorylated predominantly the histone of mol.wt. 12000. Bovine heart kinase phosphorylated several low-molecular-weight cytoplasmic proteins and no nuclear proteins. With a DEAE-cellulose column either enzyme activity could be resolved into a number of peaks. The substrate specificities of these peaks indicate that there are at least two different forms of the enzyme in each preparation of polyribosomes.

Guanosine 3’:5’-Cyclic Monophosphate -Dependent Particulate Protein Kinase Activity from Yeast (Saccharomyces cerevisiae)

1999 ◽  
Vol 54 (1-2) ◽  
pp. 84-93 ◽  
Author(s):  
Hans Eckstein ◽  
Birgit Flügge
Keyword(s):  
Protein Kinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Temperature Tolerance ◽  
Maximum Activity ◽  
Partial Purification ◽  
Protein Kinase Activity ◽  
Ph Optimum ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Concentrations

Continuing our studies on cGMP in growing yeast we detected a particulate cGMPdependent protein kinase (Pk-G), which was solubilized by detergents and NaCl. It achieves maximum activity at 25 °C and pH = 6.8, high concentrations of substrate proteins or cGMP produce saturation. Casein and histones are appropriate substrates, phosphatase-pretreated histone H-2a provokes outstandingly high activity. Pk-G differs from cAMP-dependent protein kinase (Pk-A) with respect to pH optimum, temperature tolerance above 50 °C, and stability. Partial purification is achieved by chromatography with DEAE-cellulose, Sepharose, and cGMP-substituted Sepharose. The latter step also markedly removes Pk-A. At least three proteins with Pk-G-activity and high cGMP-affinity are separated by polyacrylamide-gel-electrophoresis. Their apparent molecular masses, as deduced from comigrating marker proteins, differ considerably from those of other Pk-G’s, but also of Pk-A’s

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.

scholarly journals A hepatic soluble cyclic nucleotide-independent protein kinase. Stimulation by basic polypeptides.

1979 ◽  
Vol 254 (12) ◽  
pp. 5049-5052 ◽  
Author(s):  
M Yamamoto ◽  
W E Criss ◽  
Y Takai ◽  
H Yamamura ◽  
Y Nishizuka
Keyword(s):  
Protein Kinase ◽  
Cyclic Nucleotide ◽  
Basic Polypeptides ◽  
Independent Protein

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.

Nitric oxide and cGMP protein kinase activity in aged ventricular myocytes

2001 ◽  
Vol 281 (6) ◽  
pp. H2304-H2309 ◽  
Author(s):  
Qihang Zhang ◽  
Bruno Molino ◽  
Lin Yan ◽  
Todd Haim ◽  
Yakir Vaks ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Nitric Oxide Donor ◽  
Protein Kinase Activity ◽  
Edge Detector ◽  
Negative Effects ◽  
Dependent Protein

We tested the hypothesis that nitric oxide-induced negative functional effects through cGMP would be reduced in aged cardiac myocytes. Maximum rate of shortening ( R max) and percent shortening of ventricular myocytes from young (6 mo) and old (3 y) rabbits were studied using a video edge detector. cGMP-dependent phosphorylation was examined by electrophoresis and autoradiography. Myocytes received a nitric oxide donor S-nitroso- N-acetyl-penicillamine (SNAP, 10−7, 10−6, and 10−5 M) followed by KT-5823 (10−6 M), a cGMP protein kinase inhibitor. Baseline function was similar in young and old myocytes (89.1 ± 4.5 young vs. 86.4 ± 8.3 μm/s old R max, 5.6 ± 0.3 vs. 5.2 ± 0.7%shortening). SNAP (10−5 M) decreased R max in both young (25%, n = 6) and old myocytes (24%, n = 7). SNAP also reduced percent shortening by 28% in young and 23% in old myocytes. The negative effects of SNAP were partially reversed by KT-5823 only in young myocytes. Multiple proteins were phosphorylated by cGMP, and KT-5823 could reduce this effect. The degree of phosphorylation was significantly less in old myocytes. These results suggest that the functional response of ventricular myocytes to nitric oxide was preserved during aging. However, the importance of cGMP-dependent protein phosphorylation was decreased, indicating a shift to other pathways.

scholarly journals Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src.

1982 ◽  
Vol 2 (2) ◽  
pp. 199-206 ◽  
Author(s):  
T D Gilmore ◽  
K Radke ◽  
G S Martin
Keyword(s):  
Protein Kinase ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Defective Mutant ◽  
Sarcoma Virus

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.

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