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At least 19 recordsLinked to original sources

PubMed1979

Characterization of small cAMP-binding fragments of cAMP-dependent protein kinases.

Monomeric cAMP-binding fragments of molecular mass 16,000 and 14,000 daltons were obtained by Sephadex G-75 chromatography of partially trypsin-hydrolyzed regulatory subunits of cAMP-dependent protein kinase isozymes I and II, respectively. The Stokes radii were 19.1 and 16.4 A, the frictional ratios were 1.15 and 1.03, and the sedimentation coefficients were 1.94 and 1.91 S for the 16,000- and 14,000-dalton fragments, respectively. The 16,000-dalton fragment retained specific cyclic nucleotide binding characteristics of the native protein. The specificity of cyclic nucleotide binding to the 14,000-dalton fragment (cAMP greater than cIMP = 8-bromo-cAMP = 8-oxo-cAMP greater than cUMP = cGMP) differed from that of the native subunit (cAMP = 8-oxo-cAMP greater than 8-bromo-cAMP greater than cIMP greater than cUMP = cGMP). The 14,000-dalton fragment bound nearly 1 mol of cAMP/mol of fragment. The binding exchange rate of cAMP was much faster for the 14,000-dalton fragment than for either of the native regulatory subunits or for the 16,000 dalton fragment. Although hemin inhibited cAMP binding to the native regulatory subunits and to the 16,000 dalton fragment, the molecule did not affect cAMP binding to the 14,000-dalton fragment. Both of the native regulatory subunits and the isolated 16,000- and 14,000-dalton fragments could be covalently labeled with the photoaffinity analog, 8-N3-[32P]cAMP. The 14,000-dalton fragment could not be phosphorylated and neither fragment could recombine with the catalytic subunit to inhibit its activity. The results indicate that the functional entities of the regulatory subunit other than cAMP binding are destroyed by trypsin. The properties of the 16,000-dalton fragment suggest that the intact cAMP-binding site is contained in a small trypsin-resistant "core" of the native regulatory subunit. The properties of the 14,000-dalton fragment imply that part of the binding site of the native regulatory subunit was slighlty modified or lost during preparation of this fragment.

Animals↗

PubMed1976

Influence of adenosine, cAMP and db-cAMP on responses of the isolated terminal guinea-pig ileum to electrical stimulation.

Adenosine, cAMP and db-cAMP were found to inhibit nerve-mediated contractions of the terminal ileum elicited by electrical stimulation (ES). Responses to ES at 30 Hz, which are believed to be of adrenergic origin, were considerably more affected than the responses to ES at 3 Hz, which may be of purinergic orign. The following order of relative inhibitory potencies was established: adenosine greater than cAMP greater than db-cAMP. Adenosine, cAMP and db-cAMP did not affect the contractile responses of the preparation to histamine, potassium and noradrenaline. Therefore, the observed inhibition of nerve-mediated contractions was thought to be due to an action on presynaptic sites, i.e. on the neural network within the intestine. This action was antagonized by an increase in external calcium and by aminophylline, which is known to increase intracellular calcium. The present experiments suggests that the inhibitory action of adenosine, cAMP and db-cAMP results from a reduction of calcium fluxes across the neural membrane and to a subsequent reduction in the release of neurotransmitter(s). The inhibitory effect of cAMP appeared to be nonspecific and was presumably related to adenosine in its molecule.

Adenosine↗

PubMedDate not supplied

Human platelet aggregation and cAMP system--cAMP level, adenyl cyclase, phosphodiesterase.

The possibility of a direct and casual relationship between various parameters of the adenosine 3',5'-cyclic monophosphate (cAMP) system, e.g., the level of cAMP, adenyl cyclase activity and phosphodiesterase activity, and platelet aggregation was studied by measuring the effects of various environmental conditions, as well as metabolic inhibitors, aggregating agents and aggregation inhibitors upon these parameters. A competitive binding technique using 3H labeled cAMP was used to determine the level of cAMP in intact platelets, and a high performance liquid chromatographic method was developed to measure the adenyl cyclase and phosphodiesterase activities in the platelet membrane fraction. Although the availability of substrate adenosine triphosphate (ATP) correlated well with the amount of cAMP produced, and in turn the availability of cAMP seemed to have a direct effect upon the reversibility of shape changes induced by the various stimuli, the only effect upon aggregation concerned the extent to which it occurred. No direct correlation of the level of cAMP with either the actual inhibition or activation of the aggregation mechanism was observed.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1977

cAMP,-induced changes in cAMP-binding sites on D; discoideum amebae.

Cell surface levels of 3H-cAMP binding to Dictyostelium discoideum amebae are dramatically reduced when cells are preincubated with cAMP. This decrease in 3H-cAMP binding is shown to reflect a loss in the number of binding sites and not in any significant change in their affinity constants(s). cAMP-mediated loss of its binding sites requires the continued presence of the cyclic nucleotide and does not depend upon protein synthesis. Reapparition of sites, which occurs when cAMP is eliminated from the media, also does not depend upon protein synthesis. Experiments using metabolic inhibitors and heat-killed cells suggest that the loss of binding sites is a direct consequence of the formation of cAMP-binding protein complexes.

Azides↗

PubMed1978

Studies of cAMP metabolism in cultured hepatoma cells: presence of functional adenylate cyclase despite low cAMP content and lack of hormonal responsiveness.

The ability of isoproterenol, glucagon, PGE1 and cholera toxin to stimulate the synthesis of cAMP and protein kinase activity in line of liver cells (BRL) and a line of rat hepatoma cells (H35) has been determined. The concentration of cAMP in BRL cells (approximately 10 pmoles/mg protein) is in the range reported for other cultured cell lines but H35 cells contain extraordinarily low amounts of this cyclic nucleotide (approximately 0.05 pmoles/mg protein). Isoproterenol and PGE1 caused an increase in cAMP content, and protein kinase activation in BRL cells, although glucagon was ineffective. H35 cells, in contrast, were completely insensitive to all hormonal agonists. Despite this fact, cholera toxin was able to produce a marked increase in cAMP content, adenylate cyclase activity and protein kinase activation in H35 cells. binding studies with [125 I]-iodohydroxybenzylpindolol, a specific beta-adrenergic receptor antagonist, revealed that each H35 cell possesses fewer than 10 beta-adrenergic receptors whereas BRL cells contain 2-5,000 receptors per cell. The low level of cAMP in H35 cells appears to result from a combination of totally unstimulated adenylate cyclase and apparently elevated phosphodiesterase activities.

Adenylate Kinase↗

PubMed1979

In vivo preparation of [32P]cAMP and thin-layer chromatography of cAMP-related compounds.

A simple and rapid method for preparing [32P]adenosine 3'5'-cyclic monophosphate (cAMP) is described. A culture of an Escherichia coli mutant which excretes cAMP about 150 times faster than does a wild-type strain was incubated overnight with [32P]orthophosphate of high specific activity (e.g., 4000 Ci/mol (1 Ci = 37 GBq). The [32P]cAMP which accumulated extracellularly was then purified to 99.9% radiochemical purity in less than 4 h by adsorption to charcoal and alumina column chromatography. A two-dimensional chromatography system using a PEI-cellulose plate is also described which should prove useful for studying cAMP metabolism with 32P- or 3H-labeled cAMP or ATP.

Chromatography, Thin Layer↗

PubMed1979