scholarly journals Binding proteins for adenosine 3′:5′-cyclic monophosphate in bovine adrenal cortex

1977 ◽  
Vol 165 (3) ◽  
pp. 561-573 ◽  
Author(s):  
Stein Ove Døskeland ◽  
Per Magne Ueland
Keyword(s):  
Ionic Strength ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adrenal Cortex ◽  
Binding Proteins ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity

Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP–protein phosphotransferase, EC 2.7.1.37) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (Kd) of about 0.1μm, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K+, Mg2+ and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (Kd<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.

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.

cAMP-dependent protein kinase defines a family of enzymes

1993 ◽  
Vol 340 (1293) ◽  
pp. 315-324 ◽  
Keyword(s):  
Protein Kinase ◽  
Consensus Sequence ◽  
Protein Kinase Inhibitors ◽  
Catalytic Subunit ◽  
Affinity Binding ◽  
Dependent Protein Kinase ◽  
High Affinity Binding ◽  
High Affinity ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

The structure of the recombinant mouse catalytic subunit of cAMP-dependent protein kinase is reviewed with particular emphasis on the overall features and specific amino acids that are shared by all members of the eukaryotic protein kinase family. The crystal structure of a ternary complex containing both MgATP and a twenty-residue inhibitor peptide defines the precise role of the conserved residues that are clustered at the active site. In addition to catalysing the post-translational modification of other proteins, the catalytic subunit is itself subject to covalent modifications. It is a phosphoprotein and is also myristylated at its amino terminus. The enzyme when crystallized in the presence of detergent shows a detergent molecule bound to an acyl pocket that is presumably occupied by the myristyl moiety in the mammalian enzyme. When expressed in E.coli , the catalytic subunit is autophosphorylated at four sites. Two stable phosphates at Ser338 and Thrl97 interact with multiple protein side chains thus explaining why they are inaccessible to phosphatases. Although all substrates and inhibitors of the catalytic subunit share a general minimum consensus sequence, the high affinity binding of protein inhibitors such as the regulatory subunits and the heat stable protein kinase inhibitors require additional determinants that lie beyond the consensus site. These two physiological inhibitors of the catalytic subunit appear to use different sites to achieve high-affinity binding.

Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA

2008 ◽  
Vol 413 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Bastian Zimmermann ◽  
Sonja Schweinsberg ◽  
Stephan Drewianka ◽  
Friedrich W. Herberg
Keyword(s):  
Protein Kinase ◽  
Metal Ions ◽  
Metal Binding ◽  
Molecular Mechanisms ◽  
Regulatory Subunit ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
C Subunit ◽  
Pseudosubstrate Inhibitors

Conformational control of protein kinases is an important way of modulating catalytic activity. Crystal structures of the C (catalytic) subunit of PKA (protein kinase A) in complex with physiological inhibitors and/or nucleotides suggest a highly dynamic process switching between open and more closed conformations. To investigate the underlying molecular mechanisms, SPR (surface plasmon resonance) was used for detailed binding analyses of two physiological PKA inhibitors, PKI (heat-stable protein kinase inhibitor) and a truncated form of the R (regulatory) subunit (RIα 92–260), in the presence of various concentrations of metals and nucleotides. Interestingly, it could be demonstrated that high-affinity binding of each pseudosubstrate inhibitor was dependent only on the concentration of divalent metal ions. At low micromolar concentrations of Mg2+ with PKI, transient interaction kinetics with fast on- and off-rates were observed, whereas at high Mg2+ concentrations the off-rate was slowed down by a factor of 200. This effect could be attributed to the second, low-affinity metal-binding site in the C subunit. In contrast, when investigating the interaction of RIα 92–260 with the C subunit under the same conditions, it was shown that the association rate rather than the dissociation rate was influenced by the presence of high concentrations of Mg2+. A model is presented, where the high-affinity interaction of the C subunit with pseudosubstrate inhibitors (RIα and PKI) is dependent on the closed, catalytically inactive conformation induced by the binding of a nucleotide complex where both of the metal-binding sites are occupied.

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