An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

Inhibition of Escherichia coli growth by cyclic adenosine 3', 5'-monophosphate

Cyclic AMP inhibits growth rate of E. coli Hfr 3000. Doubling times in glucose minimal medium increased from 60 to about 90 minutes with the addition of 5 mM cAMP. This effect is specific since it was not observed when the cyclic nucleotide was replaced by 5′ AMP, ADP, ATP or adenosine. Half maximal inhibition was obtained with 1 to 3 mM cyclic AMP. This inhibition occurs only with those carbon sources which are known to decrease intracellular cyclic AMP levels, i.e. glucose and pyruvate. No inhibition was observed with succinate, malate or glycerol.     

Modulation of oocyte maturation by cyclic adenosine 3', 5'-pyrophosphate

The germinal vesicle (GV) of follicle-enclosed oocytes in mammals remains arrested at the dictyate state of meiosis. Upon releasing the oocytes from the follicles, the meiotic process resumes, leading to dissolution of the GV (GVBD), suggesting that factors in the follicular fluid sustain the meiotic arrest of oocytes. In the present study the spontaneous resumption of meiosis was blocked by the addition of cyclic adenosine 3', 5'-pyrophosphate (cAPP) plus dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP), at final concentrations of 25 and 50 microM, respectively. These compounds were ineffective when added separately at these concentrations. None of the other related compounds tested with dbcAMP blocked GVBD. Bovine follicular fluid (BFF) was analyzed for inhibitors of GVBD. BFF was extracted with 70% ethanol and the ethanolic extract chromatographed on Dowex 1-X8 column. The fraction eluted with 0.1 N HCl markedly inhibited GVBD of isolated mouse oocytes in combination with dbcAMP. The active BFF substance and cAPP block spontaneous GVBD of mouse oocytes and may be related substances. The present study supports the thesis that meiotic arrest at the dictyate stage in oocytes is sustained by factors present in follicular fluid and may act in association with cAMP.     

Compartmentation of cyclic adenosine 3',5'-monophosphate signaling in caveolae.

The cAMP-signaling pathway is composed of multiple components ranging from receptors, G proteins, and adenylyl cyclase to protein kinase A. A common view of the molecular interaction between them is that these molecules are disseminated on the plasma lipid membrane and random collide with each other to transmit signals. A limitation to this idea, however, is that a signaling cascade involving multiple components may not occur rapidly. Caveolae and their principal component, caveolin, have been implicated in transmembrane signaling, particularly in G protein-coupled signaling. We examined whether caveolin interacts with adenylyl cyclase, the membrane-bound enzyme that catalyzes the conversion of ATP to cAMP. When overexpressed in insect cells, types III, IV, and V adenylyl cyclase were localized in caveolin-enriched membrane fractions. Caveolin was coimmunoprecipitated with adenylyl cyclase in tissue homogenates and copurified with a polyhistidine-tagged form of adenylyl cyclase by Ninitrilotriacetic acid resin chromatography in insect cells, suggesting the colocalization of adenylyl cyclase and caveolin in the same microdomain. Further, the regulatory subunit of protein kinase A (RIIalpha, but not RIalpha) was also enriched in the same fraction as caveolin. Gsalpha was found in both caveolin-enriched and non-caveolin-enriched membrane fractions. Our data suggest that the cAMP-signaling cascade occurs within a restricted microdomain of the plasma membrane in a highly organized manner.     

Regulation of dimorphism in Histoplasma capsulatum by cyclic adenosine 3',5'-monophosphate.

During temperature-induced transition of the dimorphic pathogenic fungus Histoplasma capsulatum from the single yeast cell form to the multicellular mycelial form, there was an increase in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels as well as a striking accumulation of cAMP in the medium. cAMP levels also changed during the reverse mycelium-to-yeast transition.     

Regulatory functions of cyclic adenosine 3',5'-monophosphate in 1-methyladenine production by starfish follicle cells

The biosynthesis of 1-methyladenine (1-MeAde) in follicle cells of the starfish, Asterina pectinifera, occurred in response to a gonad-stimulating substance (GSS). Simultaneously with 1-MeAde production, the intracellular cAMP level immediately increased following the administration of GSS. This level in follicle cells markedly depended on GSS concentration. Although 1-MeAde production was also induced by 1-methyladenosine, it caused no increase in cAMP content. It thus appears that the effect of GSS on starfish follicle cells results in the receptor-mediated formation of cAMP.     

Effect of organotin compounds and hexachlorophene on brain adenosine cyclic 3',5'-monophosphate metabolism.

The effect of triethyltin (TET), triphenyltin (TPT), hexachlorophene (HCP) and cuprizone on adenosine cyclic 3',5'-monophosphate (cyclic AMP) production in rat brain was examined both in vitro and in vivo. TET and TPT inhibited basal adenylate cyclase activity of brain homogenate at a concentration as low as 1 microM in vitro but these compounds had no effect on norepinephrine (NE) and dopamine(DA)-stimluated enzyme activity. HCP and cuprizone failed to inhibit adenylate cyclase activity. In vivo TET given intravenously at a dose rate of 10 mg/kg decreased the cyclic AMP content of cerebrum, but not of medulla. TPT and HCP give intravenously and intraperitoneally respectively failed to decrease the cyclic AMP content of the cerebrum. In the case of TET the reduction in cyclic AMP content of the cerebrum was prevented by maintaining the rats normothermic after treatment. On the basis of these results the inhibition of adenylate cyclase produced by TET in brain homogenates in vitro would not appear to be involved in the development of nervous changes associated with acute TET toxicity, or in the production of progressive brain oedema caused by TET, HCP and cuprizone.     

Linear velocity of cyclic adenosine 3',5'-monophosphate transport in corn coleoptiles

The transport of cyclic adenosine 3′, 5′-monophosphate in corn coleoptile segments is very rapid. The linear velocity of basipetal transport is 183 millimeters per hour, while the velocity of acropetal transport is 79 millimeters per hour. Transport velocity as well as intensity thus appear to be polar in the corn coleoptile. Application of metabolic inhibitors such as cyanide, ouabain, and 2,4-dinitrophenol increase rather than decrease the velocity and intensity of transport. The mechanism of transport in light of these data is discussed.