Cyclic AMP Binding Sites Research Articles

The binding of cyclic nucleotides to membranes of the endoplasmic reticulum of normal and neoplastic rat liver

Studies are presented which demonstrate that the smooth and rough endoplasmic membranes of normal and neoplastic rat liver possess binding sites for cyclic nucleotides exhibiting a high degree of specificity. In contrast to normal liver, which has only a single binding site for cyclic AMP on membranes of the endoplasmic reticulum, cyclic AMP binding to the intracellular membranes of hepatoma 5123C and 7777 exhibits two apparent binding sites. The binding constant for cyclic AMP of one site on the tumor membranes is comparable to that of the normal liver, whereas the value of the second intrinsic association constant is 4- to 40-fold greater than liver. The possibility that the presence of the second cyclic AMP binding site might be a function of the rapid growth of the tumors was unlikely since membrane preparations from neonatal rats showed a single affinity association constant which was similar to that of normal liver. In addition, membranes of the endoplasmic reticulum of the Morris hepatomas 5123C and 7777 exhibit a binding site for cyclic GMP which is absent from the intracellular membranes of liver.

Some biochemical properties of alkyl phosphotriesters of cyclic AMP

The biochemical properties of several alkyl phosphotriesters of cyclic AMP were studied with respect to their interactions with beef heart protein kinase and cyclic nucleotide phosphodiesterase. Ethyl and propyl triesters did not enhance the phosphorylation of histone by protein kinase and methyl, ethyl, propyl and butyl triesters were poor competitors for the cyclic AMP binding site of the enzyme. However, these alkyl phosphotriesters were effective inhibitors of cyclic nucleotide phosphodiesterase with the K i 's arrayed in the following order: methyl > ethyl > propyl > butyl > cetyl triester. Metabolic studies with mice indicated that intraperitoneal injection of low doses of propyl triester for one week significantly increased cyclic AMP concentration.

Adenosine 3':5'-monophosphate-regulated phosphorylation of erythrocyte membrane proteins. Separation of membrane-associated cyclic adenosine 3':5'-monophosphate-dependent protein kinase from its endogenous substrates

An adenosine 3':5'-monophosphate (cyclic AMP)-binding protein in the human erythrocyte plasma membrane was isotopically labeled using a photoaffinity analog of cyclic AMP, N6-(ethyl 2-diazomalonyl) cyclic [3H]AMP. The cyclic AMP-binding site is located in a polypeptide chain having a molecular weight of 48,000. Cyclic AMP-binding protein and cyclic AMP-dependent protein kinase were solubilized with 0.5% Triton X-100 in 56 mM sodium borate, pH 8, but 32P-labeled membrane phosphoproteins were retained in the Triton-insoluble fraction, suggesting that the membrane-associated binding protein is not a primary substrate for protein kinase. Triton-solubilized and membrane-associated protein kinase activities were stimulated 15- and 17-fold by cyclic AMP, suggesting that the degree of association between the catalytic anc cyclic AMP-binding components was very similar in both preparations. Fractionation and characterization of membrane phosphoproteins have shown that protein III and a co-migrating minor protein are substrates for protein kinase but membrane sialoglycoproteins are not phosphorylated.

Activation of protein kinase by physiological concentrations of cyclic AMP.

When determined under the usual conditions of an excess of ligand over protein, the concentration of cyclic AMP necessary to activate pure preparations of cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:-protein protein phosphotransferase) half-maximally is in the range of 0.2-0.3 muM when casein or glycogen synthetase is used as the substrate, i.e., essentially the same as the concentration of the nucleotide that is found in resting skeletal muscle. The apparent dissociation constant for cyclic AMP bound to the protein kinase is also about 0.2-0.3 muM when measured under similar conditions. The concentration of the protein kinase in muscle is relatively high (0.23 muM), however, and under these conditions the apparent activation constant of the enzyme for cyclic AMP is raised so that an increase in cyclic AMP levels in the tissue would cause a concomitant increase in protein kinase activity over a wide range of nucleotide concentration. As a result, it is unnecessary to invoke compartmentalization of cyclic AMP to explain how it can control protein kinase activity in vivo. Another factor that may increase the effectiveness of changes in cyclic AMP concentration is the heat-stable protein inhibitor of protein kinase that may function to inhibit the activity of nearly all the protein kinase catalytic subunit dissociated by basal concentrations of cyclic AMP. Finally, the near equity between the concentration of cyclic AMP binding sites and the ligand itself provides a potential mechanism whereby agents can affect the total cyclic AMP content without directly affecting adenylate cyclase, cyclic AMP phosphodiesterase, or cyclic AMP transport.

Short-term binding and hydrolysis of cyclic 3':5'-adenosine monophosphate by aggregating Dictyostelium cells.

Transient binding of cyclic AMP to aggregating cells of Dictyostelium discoideum was measured under cyclic GMP excess in order to inhibit cyclic AMP hydrolysis by cell-bound phosphodiesterase. Cyclic GMP extended the period of half-maximal cyclic AMP binding from less than 5 sec to 1-2 min. With the same time course as bound cyclic AMP was released from the cells, labeled 5'-AMP appeared in the medium. Specificity, kinetics, and developmental regulation suggest that the cyclic AMP-binding sites exposed in living cells are identical with receptor sites for the chemotactic response. The functioning of the cyclic AMP-receptor/phosphodiesterase system and its formal similarity with the synaptic acetylcholine-receptor/esterase system are discussed.

Cyclic-AMP binding sites on the cell surface of thymic lymphocytes

Exogenous cyclic-AMP binds to intact thymic lymphocytes in a linear manner over the mitogenic concentration range 2.5 × 10 −8 M to 2.5 × 10 −7 M, giving an apparent binding constant of 5 × 10 −8 M. The same concentrations of cyclic-GMP and 5'-AMP are non-mitogenic and do not bind appreciably. The cyclic-AMP once bound cannot be displaced by addition of excess cyclic-AMP, cyclic-GMP or 5'-AMP. The bound cyclic-AMP may be released at 37°C and recovered quantitatively as cycliv-AMP in the incubation medium. These observations are consistent with the possibility that thymic lymphocytes have cyclic-AMP binding sites on their surface.

Photo-affinity labels for adenosine 3':5'-cyclic monophosphate.

Three derivatives of cyclic AMP that are potentially useful as photo-affinity labels were synthesized. One of these derivatives is, upon photolysis, specifically incorporated into the cyclic AMP-binding site of rabbit muscle phosphofructokinase. The use of these derivatives in isolating and identifying cyclic AMP receptor sites is discussed.