scholarly journals Differential activation of type-I and type-II adenosine 3′:5′-cyclic monophosphate-dependent protein kinases in liver of glucagon-treated rats

1978 ◽  
Vol 170 (3) ◽  
pp. 469-477 ◽  
Author(s):  
G Schwoch
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Deae Cellulose ◽  
Type I ◽  
Type Ii ◽  
Cytosolic Protein ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Independent Protein

The protein-bound cyclic AMP and the activity of cytosolic protein kinases in the presence and absence of cyclic AMP were determined in rat liver up to 2h after injection of glucagon. On the basis of the different salt-sensitivities of the activated cyclic AMP-dependent proteinkinases I and II, an activation of protein kinase II restricted to the high cyclic AMP concentrations present in the first 30 min after hormone injection was found. Essentially the same result was obtained by chromatographic analysis on DEAE-cellulose of liver cytosol from untreated rats and from rats killed at 2 and 60 min after glucagon injection. Protein kinase II activation was only detected at 2 min after injection. In contrast, the cyclic AMP-dependent protein kinase I was found to be nearly totally activated at 2 min and to be still almost as active at 60 min after the hormone stimulus, whereas the amount of bound cyclic AMP and the activation of total cytosolic protein kinases had fallen to two-thirds of their maximal values during this time period. A third cyclic AMP-independent protein kinase, which co-chromatographed with protein kinase type II, could be clearly distinguished from the two cyclic AMP-dependent kinases by use of the heat-stable inhibitor from bovine muscle, which totally inhibited the cyclic AMP-dependent enzymes, but stimulated the cyclic AMP-independent protein kinase.

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 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 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.

scholarly journals Isolation of two myosin light-chain kinases from bovine carotid artery and their regulation by phosphorylation mediated by cyclic AMP-dependent protein kinase

1982 ◽  
Vol 203 (3) ◽  
pp. 583-592 ◽  
Author(s):  
Ramesh C. Bhalla ◽  
Ram V. Sharma ◽  
Ramesh C. Gupta
Keyword(s):  
Protein Kinase ◽  
Carotid Artery ◽  
Cyclic Amp ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Type I ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Myosin light-chain kinase was purifed from bovine carotid artery. Approx. 90% of myosin kinase was extracted in the supernatant fraction with buffer containing EDTA during myofibril preparation. The soluble fraction yielded two distinct peaks on DEAE-Sephacel chromatography. Peak I was eluted at a conductance of 11–12mmho and was completely dependent on Ca2+–calmodulin for its activity. Peak II was eluted at a conductance of 13–14mmho and showed approx. 15% Ca2+-independent activity. The myosin kinases I and II were further purified by affinity chromatography by using calmodulin coupled to Sepharose 4B, which resulted in 960-and 650-fold purification of type I and type II kinases respectively. Myosin kinase II activity was completely Ca2+-dependent after affinity chromatography on the calmodulin–Sepharose column. Myosin kinases I and II were phosphorylated by cyclic AMP-dependent protein kinase. In the presence of bound calmodulin 0.5–0.7mol of phosphate was incorporated/mol of myosin kinases I and II. On the other hand, in the absence of bound calmodulin 1–1.4mol of phosphate was incorporated/mol of kinases I and II. Phosphorylation in the absence of calmodulin significantly decreased the myosin kinase activity of both enzymes, and the decrease in myosin kinase activity was due to a 3–5-fold increase in the amount of calmodulin required for half-maximal stimulation of both type I and type II kinases. The regulation of myosin kinase activity by cyclic AMP-dependent phosphorylation would suggest that β-adrenergic-mediated relaxation of vascular smooth muscle may be partly due to the direct interaction of cyclic AMP at the site of contractile proteins.

Protein Kinases in HeLa Cells and in Human Cervix Carcinoma

1981 ◽  
Vol 36 (7-8) ◽  
pp. 552-561 ◽  
Author(s):  
W. Pyerin ◽  
N. Baibach ◽  
D. Kübler ◽  
V. Kinzel
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Hela Cells ◽  
Deae Cellulose ◽  
Type I ◽  
Cervix Carcinoma ◽  
Ionic Detergent ◽  
Membrane Bound

Abstract Extracts of HeLa cell fractions were analyzed by DEAE-and phospho-cellulose chromatog­ raphy for their range of cyclic AMP-dependent and -independent protein kinase activities phosphorylating histone and/or phosvitin; extractions were by phosphate buffered saline (soluble protein kinases) and the non-ionic detergent NP-40 (membrane-bound protein kinases). The soluble fraction contained (i) cyclic AMP-dependent histone kinases type I and II as evidenced by their behaviour on DEAE-cellulose and inhibition by the specific heat-and acid-stable protein kinase inhibitor (PKI) in a dose-related manner; both types I and II as well as their purified catalytic subunit also phosphorylated protamine and — with very low efficiency -casein but not phosvitin; (ii) a histone kinase (H), insensitive to cyclic AMP and PKI, also accepting protamine as substrate but not either casein or phosvitin; (iii) a phosvitin kinase (P), insensitive to cyclic AMP and PKI, which also phosphorylates casein but not histone or protamine. These four enzyme species were also found in NP-40 extracts of 27000x g residues which, however, contained further histone and phosvitin kinase activities as yet unspecified. NP-40 extracts of the microsomal fraction possessed, besides unspecified histone and phosvitin kinase activity, only the phosvitin kinase P and appeared to be devoid of histone kinases I, II, and H. The occurrence and ratios of the protein kinases classified suggest an ordered distribution over the diverse subcellular fractions of HeLa cells. The overall pattern of soluble and membrane-bound histone and phosvitin kinases in extracts of cervix carcinoma tissue, the in vivo correlate of HeLa cells, closely resembled that of similar extracts of HeLa cells. HeLa cells hence appear, despite their long in vitro history, to express protein kinase activities similar to those of their in vivo ancestors, recommending them as a subject for the study of (certain) human protein kinase systems.

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