Cyclic AMP Binding Activity Research Articles

The cyclic AMP receptor of Escherichia coli: Immunological studies in extracts of Escherichia coli and other organisms

An antiserum specific for the cyclic adenosine 3′,5′-monophosphate receptor from Escherichia coli has been employed to detect the presence of a similar protein in cellular extracts of a number of diverse organisms. In Ouchterlony double-diffusion experiments cellular extracts from Photobacterium fisheri, Aerobacter aerogenes, Proteus mirabilis, and Salmonella typhimurium all showed precipitin bands with E. coli cyclic AMP receptor-antiserum. The extract from Caulobacter crescentus exhibited slight cross-reactivity. Similar results were obtained with an immuno-precipitation assay used to quantitate the amount of cyclic AMP receptor-like protein present. Extracts from a variety of organisms were found to bind cyclic AMP when the usual (NH 4) 2SO 4 precipitation assay for cyclic AMP receptor was employed. Only the extract from Methanosarcina barkeri was inactive. Some extracts prepared from E. coli grown on Luria broth were observed to have no cyclic AMP binding activity. Antiserum was used to determine the presence of cyclic AMP receptor in these inactive extracts. These preparations usually regain binding activity on standing at 4°C for 2–3 days.

Adenosine Cyclic 3′:5′-Monophosphate-dependent Protein Kinase from Rabbit Erythrocytes: PURIFICATION AND CHARACTERIZATION OF MULTIPLE FORMS

Abstract Adenosine cyclic 3':5'-monophosphate (cyclic AMP)-dependent protein kinase from rabbit erythrocytes has been resolved into three active fractions possessing both phosphotransferase and cyclic AMP-binding activities. These enzymes are arbitrarily designated as kinases I, IIa, and IIb. The kinetic properties and substrate specificities of these kinases are remarkably similar. All three variants catalyze the phosphorylation of calf thymus histones and are stimulated by low concentrations of cyclic AMP. The molecular weights of these enzymes have been estimated by gel filtration to be 170,000, 120,000, and 240,000, corresponding to kinases I, IIa, and IIb, respectively. The three kinases differ in temperature (53°) stability. Kinases IIa and IIb are inactivated more slowly than kinase I. However, the rate of inactivation of kinase I is greatly diminished in the presence of both ATP and Mg2+. A similar protection by these compounds is not observed with kinases IIa and IIb. Each of these kinases appears to be constructed of two dissimilar functional subunits: a catalytic subunit and a regulatory subunit. Cyclic AMP causes the two dissimilar functional subunits to dissociate by binding to the regulatory moiety. A similar activation may be accomplished by protamine. In addition, protamine enhances the binding of cyclic AMP to the kinases as determined by the Millipore filtration assay. The regulatory moiety of kinase I cross-reacts with the catalytic moiety of both kinases IIa and IIb.

Mechanism of action of gonadotropin. 3. Binding of adenosine 3',5'-monophosphate by the 105,000 X g supernatant fraction from homogenates of calf ovaries.

The binding of adenosine 3′,5′-monophosphate (cyclic AMP) to calf ovary cytosol has been investigated by equilibrium dialysis, membrane filter assay, and sucrose density gradient analysis. Protein purified from calf ovary cytosol bound cyclic AMP with an apparent dissociation constant kd of 4.1 × 10-9M. The cyclic AMP-binding protein complex exhibited a sedimentation coefficient of approximately 4S. The cyclic AMP-binding activity was inactivated by pronase, heat treatment, and by the addition of 0.1% SDS. The specificity of binding for cyclic AMP was tested by the addition of a variety of nucleotides to the reaction mixture and their ability to compete with cyclic [3H]AMP for the protein binding sites. At a concentration of 5 × 10-7M, only cyclic AMP competed effectively with cyclic [3H]AMP. At higher concentrations (2.5 × 10-5M), dibutyryl cyclic AMP, cyclic GMP, cyclic CMP, and cyclic UMP were able to displace cyclic [3H]AMP from the binding protein. Purification of the cyclic AMP-binding protein by DE...

Cyclic Adenosine 3′,5′-Monophosphate-dependent Protein Kinase of Human Erythrocyte Membranes

Abstract A cyclic AMP (adenosine 3', 5'-monophosphate)-dependent protein kinase has been found in membranes derived from human erythrocytes which accounts for greater than 70% of the total cyclic AMP-dependent protein kinase activity of these cells. Components of the erythrocyte membranes were phosphorylated by membrane-bound protein kinase, but stimulation of the rate of phosphorylation by cyclic AMP was observed only when histones or protamine served as substrates. The membrane-associated cyclic AMP-dependent protein kinase resembles many soluble protein kinases; it requires 20 mm Mg2+ for maximal activity, has apparent Km values of 26 µg per ml and 8.3 µm for protamine and ATP, respectively, and a Ka for cyclic AMP of 28 nm. Erythrocyte membranes specifically bind cyclic AMP and exhibit a dissociation constant of 3.3 nm for the membrane-cyclic AMP complex. Cyclic AMP-independent protein kinase activity was dissociated from the membrane by treatment with 1 m NH4Cl, but the cyclic AMP-binding activity was retained by the particulate fraction. These findings suggest that the cyclic AMP-binding moiety is firmly integrated into the membrane structure, whereas the catalytic moiety may only be loosely associated with the membrane.

Role of Cyclic Adenosine 3',5'-Monophosphate in the Induction of Hepatic Enzymes: III. INTERACTION OF HYDROCORTISONE AND N6,O2'-DIBUTYRYL CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE IN THE INDUCTION OF TYROSINE AMINOTRANSFERASE IN CULTURED H-4-11-E HEPATOMA CELLS

The induction of tyrosine aminotransferase in H-4-11-E hepatoma cells (a strain derived from H-35 Reuber hepatoma) in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (cyclic AMP) requires low concentrations of hydrocortisone. Incubation of the cells with 10-4 m dibutyryl cyclic AMP alone failed to induce the enzyme. Hydrocortisone must be continuously present for the induction of tyrosine aminotransferase in the presence of dibutyryl cyclic AMP. The induction of tyrosine aminotransferase by 10-8 m hydrocortisone plus 10-4 m dibutyryl cyclic AMP requires both protein and RNA synthesis. Glucagon (free of insulin) alone or with hydrocortisone neither increases the adenyl cyclase activity nor induces tyrosine aminotransferase in H-4-11-E hepatoma cells. Hydrocortisone has no effect on the uptake of exogenous dibutyryl cyclic AMP and, conversely, dibutyryl cyclic AMP has no effect on the uptake of hydrocortisone. That hydrocortisone might increase the cyclic AMP-binding activity of soluble proteins has also been excluded. Hydrocortisone increases both the total activity of protein phosphokinase (or phosphokinases) of the cells and the phosphorylation in vivo of nuclear histones.

Further studies on the properties of the rabbit reticulocyte adenosine 3′,5′-cyclic monophosphate-dependent protein kinase

The properties of cyclic AMP-dependent protein kinase I isolated from rabbit reticulocytes were further investigated. The enzyme catalyzes the phosphorylation of histone in the presence of ATP and Mg 2+ and this reaction is stimulated by cyclic AMP. The pH optimum of the reaction was between 8.5 and 9.0, when assayed in the presence of cyclic AMP. No distinct pH optimum was observed in the absence of the cyclic nucleotide. The K m values for ATP appeared to be very similar whether it was determined in the presence ( K m = 1.7 × 10 −4 m) or absence ( K m = 2.5 × 10 −4 m) of cyclic AMP. The rate of heat inactivation of the catalytic activity and the cyclic AMP binding activity of kinase I were found to be dependent on the presence of Mg 2+, ATP, and/or cyclic AMP. In the presence of cyclic AMP, the rate of inactivation of the catalytic activity of kinase I at 53 ° was accelerated. On the other hand, the cyclic AMP binding activity appeared to be protected from heat inactivation by the cyclic nucleotide. When both ATP and Mg 2+ were present in the heating mixture, no loss of catalytic and binding activities of kinase I were observed even up to 8 min of heating at 53 °. The cyclic AMP binding activity of kinase I was almost completely inhibited by mercuric acetate at a concentration of 1 m m, while the loss in catalytic activity was only 50%. These results substantiate our previous observation that kinase I contains two nonidentical subunits, a catalytic subunit and a cyclic AMP binding subunit.