Low Mr GTP-binding proteins in human platelets: Cyclic AMP-dependent protein kinase phosphorylates m22KG(I) in membrane but not c21KG in cytosol

1989 ◽  
Vol 160 (1) ◽  
pp. 235-242 ◽  
Author(s):  
Koh-ichi Nagata ◽  
Seiji Nagao ◽  
Yoshinori Nozawa
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Binding Proteins ◽  
Human Platelets ◽  
Dependent Protein Kinase ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Dependent Protein

scholarly journals Photoaffinity labelling of cyclic GMP-binding proteins in human platelets

1993 ◽  
Vol 294 (2) ◽  
pp. 329-333 ◽  
Author(s):  
K M Tang ◽  
J L Sherwood ◽  
R J Haslam
Keyword(s):  
Protein Kinase ◽  
Cyclic Gmp ◽  
Binding Proteins ◽  
Protein S ◽  
Human Platelets ◽  
Dependent Protein Kinase ◽  
Camp Phosphodiesterase ◽  
Proteolytic Fragment ◽  
Photoaffinity Labelling ◽  
Dependent Protein

The photoaffinity labelling of platelet cyclic GMP (cGMP)-binding proteins by [32P]cGMP was studied; at least five labelled proteins (110, 80, 55, 49 and 38 kDa) were detected in platelet cytosol and four (80, 65, 49 and 38 kDa) in platelet membranes. The 110 kDa species was identified as cGMP-inhibited cyclic AMP (cAMP) phosphodiesterase (PDE III) by immunoprecipitation and by the inhibition of photolabelling by specific inhibitors of this enzyme. Similarly, the 80 kDa species was identified as cGMP-dependent protein kinase by immunoprecipitation and by the effects of cGMP analogues on photolabelling. Addition of cAMP greatly enhanced the labelling of this 80 kDa protein, implying the existence of a potentially important interaction between the effects of cGMP and cAMP. The 65 kDa photolabelled protein appears to be a novel platelet cyclic-nucleotide-binding protein. In contrast, the 49 and 55 kDa photolabelled species are probably the RI and RII regulatory subunits of cAMP-dependent protein kinase, and the 38 kDa protein(s) may be proteolytic fragment(s) of RI and/or RII.

scholarly journals Adenosine 3':5'-cyclic monophosphate-binding proteins in bovine and rat tissues

1976 ◽  
Vol 159 (2) ◽  
pp. 423-427 ◽  
Author(s):  
P H Sugden ◽  
J D Corbin
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Binding Proteins ◽  
Binding Protein ◽  
Bovine Liver ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Trypsin Treatment ◽  
Dependent Protein Kinase ◽  
Dependent Protein

1. At least two classes of high-affinity cyclic AMP-binding proteins have been identified: those derived from cyclic AMP-dependent protein kinases (regulatory subunits) and those that bind a wide range of adenine analogues (adenine analogue-binding proteins). 2. In fresh-tissue extracts, regulatory subunits could be further subdivided into ‘type I’ or ‘type II’ depending on whether they were derived from ‘type I’ or ‘type II’ protein kinase [see Corbin et al. (1975) J. Biol. Chem. 250, 218-225]. 3. The adenine analogue-binding protein was detected in crude tissue supernatant fractions of bovine and rat liver. It differed from the regulatory subunit of cyclic AMP-dependent protein kinase in many of its properties. Under the conditions of assay used, the protein accounted for about 45% of the binding of cyclic AMP to bovine liver supernatants. 4. The adenine analogue-binding protein from bovine liver was partially purified by DEAE-cellulose and Sepharose 6B chromatography. It had mol.wt. 185000 and was trypsin-sensitive. As shown by competition and direct binding experiments, it bound adenosine and AMP in addition to cyclic AMP. At intracellular concentrations of adenine nucleotides, binding of cyclic AMP was essentially completely inhibited in vitro. Adenosine binding was inhibited by only 30% under similar conditions. 5. Rat tissues were examined for the presence of the adenine analogue-binding protein, and, of those examined (adipose tissue, heart, brain, testis, kidney and liver), significant amounts were only found in the liver. The possible physiological role of the adenine analogue-binding protein is discussed. 6. Because the adenine analogue-binding protein or other cyclic AMP-binding proteins in tissues may be products of partial proteolysis of the regulatory subunit of cyclic AMP-dependent protein kinase, the effects of trypsin and aging on partially purified protein kinase and its regulatory subunit from bovine liver were investigated. In all studies, the effects of trypsin and aging were similar. 7. In fresh preparations, the cyclic AMP-dependent protein kinase had mol.wt. 150000. Trypsin treatment converted it into a form of mol.wt 79500. 8. The regulatory subunit of the protein kinase had mol.wt. 87000. It would reassociate with and inhibit the catalytic subunit of the enzyme. Trypsin treatment of the regulatory subunit produced a species of mol.wt. 35500 which bound cyclic AMP but did not reassociate with the catalytic subunit. Trypsin treatment of the protein kinase and dissociation of the product by cyclic AMP produced a regulatory subunit of mol.wt. 46500 which reassociated with the catalytic subunit. 9. These results may be explained by at least two trypsin-sensitive sites on the regulatory subunit. A model for the effects of trypsin is described.

scholarly journals Characterization of Sp-5,6-dichloro-1-β-d-ribofuranosylbenzimidazole- 3′,5′-monophosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells

1991 ◽  
Vol 279 (2) ◽  
pp. 521-527 ◽  
Author(s):  
M Sandberg ◽  
E Butt ◽  
C Nolte ◽  
L Fischer ◽  
M Halbrügge ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Prostaglandin E1 ◽  
Human Platelets ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Cell Extracts ◽  
Platelet Membranes ◽  
Camp Analogue ◽  
Dependent Protein

A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole-3′,5′-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6- DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.

scholarly journals Cyclic AMP-binding and cyclic AMP-dependent protein kinase activities in the cytosol of differentiating bone marrow erythroblasts

1981 ◽  
Vol 196 (3) ◽  
pp. 893-897 ◽  
Author(s):  
M S Setchenska ◽  
J G Vassileva-Popova ◽  
H R Arnstein
Keyword(s):  
Bone Marrow ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Binding Proteins ◽  
Kinase Activity ◽  
Binding Capacity ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Dividing Cells ◽  
Dependent Protein

Cytosolic cyclic AMP-binding capacity and cyclic AMP-dependent protein kinase activity have been studied in relation to differentiation and maturation of rabbit bone marrow erythroblasts. Using cells fractionated by velocity sedimentation at unit gravity, it was found that both activities decreased in dividing cells when calculated in terms of cell number but remained constant per cell volume. After the final cell division, cyclic AMP-dependent protein kinase activity did not change further, whereas cyclic AMP-binding capacity declined. There were no qualitative, but only quantitative, changes in the cyclic AMP-binding proteins that are present in the cytosol of developing erythroblasts. In the immature cells, the apparent KD for the interaction of binding proteins with cyclic AMP was 4 } 10(-8) M. The data suggest that changes in cyclic AMP-binding activity during differentiation of erythroid cells are due both to changes in the amount of binding proteins and in their affinity for cyclic AMP. Plasma membranes of erythroblasts were also able to bind cyclic AMP but only in dividing cells.

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