Synthesis and release of adenosine 3′: 5′-cyclic monophosphate by Chlamydomonas reinhardtii

One of the labeled compounds synthesized by Chlamydomonas reinhardtii when ³²Pi was supplied was isolated from both the cells and the medium in which the cells had grown. This compound copurified with authentic [8-³H]cAMP by TLC to a constant ratio of ³²P/³H. The compound was degraded by beef heart cyclic nucleotide phosphodiesterase to a product which cochromatographed with authentic 5′AMP, at the same rate as the hydrolysis of authentic cAMP-[³H] to 5′AMP-[³H]. In both cases, 1-Me-3-isoBu-xanthine, a specific inhibitor of the phosphodiesterase, totally blocked the reaction. It is concluded that the compound synthesized by C. reinhardtii was cAMP, 85% of which was released into the medium.     

Cyclic adenosine 3':5'-monophosphate in axenic rye grass endosperm cell cultures

Cyclic adenosine 3′:5′-monophosphate (cAMP) was extensively purified from rye grass (Lolium multiflorum) endosperm cells grown in axenic suspension culture. The cAMP was purified by neutral alumina and anion and cation exchange chromatography. The cAMP was quantitated by means of a radiochemical saturation assay using a beef heart cAMP-binding protein and also by an assay involving activation of beef heart protein kinase. The cAMP levels found (corrected for recovery of tracer cyclic 3′,5′-[8-³H]AMP included from the point of sample extraction) ranged from 2 to 12 pmol/g fresh weight. The material purified from rye grass cultures was indistinguishable from authentic cAMP with respect to chromatography in two cellulose thin layer systems, behavior on dilution in both the saturation and protein kinase activation assays, and rates of degradation by a mammalian cAMP phosphodiesterase. The cAMP from rye grass cultures was completely degraded by a mammalian cAMP phosphodiesterase, and 1-methyl-3-isobutylxanthine inhibited such degradation. The protein kinase activation and saturation assays gave essentially the same values for the cAMP content of axenic rye grass culture extracts. Material satisfying the above criteria for identity with cAMP was also isolated from the culture medium. The increase observed in medium cAMP levels during culture growth provides evidence for the synthesis and secretion of cAMP by rye grass endosperm cells in suspension culture.     

Cyclic adenosine 3':5'-monophosphate in moss protonema: a comparison of its levels by protein kinase and gilman assays

From the protonema of the moss Funaria hygrometrica (L.) Sibth, a factor indistinguishable from cyclic adenosine 3′:5′-monophosphate (cAMP) has been isolated. The factor stimulated the activity of protein kinase from rabbit skeletal muscle and co-chromatographed with authentic cAMP in two solvent systems. Its ability to stimulate protein kinase activity was completely abolished by 3′:5′-cyclic nucleotide phosphodiesterase, the rate of inactivation being similar to that of authentic cAMP. Based on these properties, this factor is identified as 3′,5′-cAMP. Cyclic AMP could be readily removed from the cells and washing the cells with water reduced the endogenous level of cAMP by 2- to 3-fold. A comparison of cAMP levels by protein kinase and Gilman assays was made. The intracellular levels determined by protein kinase assay were about 7-fold lower than the values obtained by Gilman assay. This discrepancy was due to the presence of unidentified compounds which were completely degraded by 3′:5′-cyclic nucleotide phosphodiesterase. Although these displaced labeled cAMP in the Gilman assay, they did not stimulate the protein kinase activity. The protonema may contain cyclic nucleotides other than cAMP; these will not be detected in the protein kinase assay due to the specificity of this reaction. The crude extracts were found to be unsuitable for assaying cAMP by either method.     

On the presence of adenosine 3′, 5′ -cyclic monophosphate in moss (Funariahygrometrica)

Partially purified nucleotide fraction of moss containing [14C]-labelled putative adenosine 3′, 5′ -cyclic monophosphate (cAMP) and marker authentic [3H] -cAMP was characterized by chemical deamination and also by the enzymatic hydrolysis with beef heart cyclic nucleotide phosphodiesterase. A significant conversion of marker authentic [3H] -cAMP into [3H] -inosine 3′, 5′ -cyclic monophosphate (cIMP) and [3H] -5′ adenosine monophosphate was observed by respective treatments. In contrast, the [14C] -labelled putative cAMP from control and theophylline-treated moss tissue was insensitive to chemical deamination and enzymatic hydrolysis. Apparently, the [14C] -labelled product which comigrates with authentic [3H] -cAMP does not represent true cAMP. Both the methods employed for characterization of the labelled putative cAMP were sensitive enough to detect picomole quantities of authentic [3H] -cAMP. Lack of detectability of prelabelled [14C] -cAMP in our preparations implies that the tissue may contain authentic cyclic AMP below the picomole levels. Thus, the attributed physiological role to adenosine 3′, 5′ -cyclic monophosphate in moss tissue appears somewhat skeptical.     

Identification of 3',5'-cyclic AMP in axenic cultures of Lemna paucicostata by high-performance liquid chromatography.

Nucleotides were purified from the extract of aseptically grown Lemna paucicostata 6746 by low-pressure column chromatography and thin-layer chromatography. In this partially purified sample, a compound has been identified, using a highly sensitive HPLC-fluorometric detection method, with chromatographic properties and substrate specificity for cyclic nucleotide phosphodiesterase similar to authentic 3',5'-cyclic AMP. The level of cAMP has been estimated to be 70-80 pmol/g fresh wt.     

An assay for adenyl cyclase based on a combination of sodium borate electrophoresis and paper chromatography.

We describe a method for the assay of adenyl cyclase in whole tissue homogenates. Adenosine 3′:5′-cyclic monophosphate (cAMP) formed from α-³²P-, ¹⁴C- or ³H-labeled adenosine 5′-triphosphate (ATP) substrate is isolated from all known ATP metabolites and an unknown metabolite by electrophoresis in 1% sodium borate for 40 min, followed by overnight descending chromatography in 95% ethanol:1 m ammonium acetate (70:30). The purity of the cAMP isolated is established by chromatographic techniques as well as by utilizing a purified cyclic nucleotide phosphodiesterase. The method described here also makes possible the measurement of phosphodiesterase activity in homogenates. It is rapid enough to allow routine assay of 180 samples per day, although the number of samples processed depends on the number of electrophoretic and chromatographic units available.     

Studies on the presence of adenosine cyclic 3':5'-monophosphate in oat coleoptiles

The incorporation of adenosine-8-¹⁴C into adenosine cyclic 3′:5′-monophosphate in coleoptile-first leaf segments of Avena sativa L. was investigated. Homogenates of segments incubated in adenosine-8-¹⁴C for either 4 or 10 hours were partially purified by thin layer chromatography followed by paper electrophoresis. A radioactive fraction, less than 0.06% of the ¹⁴C present in the original homogenate, migrated as adenosine cyclic 3′:5′-monophosphate during electrophoresis. Upon treatment with cyclic nucleotide phosphodiesterase, however, less than 10% of this radioactive fraction appeared as 5′-AMP. Deamination with NaNO₂ as well as further chromatographical purification also suggested that only a small fraction of the ¹⁴C in the partially purified samples could be in adenosine cyclic 3′:5′-monophosphate. The data suggest that levels of this nucleotide can probably be no greater than 7 to 11 picomoles per gram of fresh weight in oat coleoptiles. Treatment of such coleoptiles with physiologically active concentrations of indoleacetic acid, furthermore, had no significant effect on the ¹⁴C radioactivity in marker adenosine cyclic 3′:5′-monophosphate-containing fractions at any stage of purification during several experiments.     

Cyclic AMP,cyclic GMP,and bean rust uredospore germlings

Ten millimolar cyclic AMP (cAMP) or cyclic GMP (cGMP) induced bean rust uredospore germlings to undergo one round of mitosis and to form septa, processes normally associated with appressorium formation. To assess the possibility of cyclic nucleotide regulation of bean rust development, we used an 8-azido-[³²P]cAMP photoaffinity probe to identify three cyclic nucleotide binding peptides. The peptides bound either cAMP or cGMP. The phosphorylation of one peptide in uredospore germling extracts by [γ-³²P]ATP was stimulated by either 1 μM cAMP or cGMP, but only in the presence of 10 mM Na₂MoO₄, a phosphatase inhibitor. Uredospores contain about 1500 and 23 pmol cAMP and cGMP/g dry wt, respectively, as determined by radiobinding assays.     

Failure to Detect Cyclic 3', 5'-Adenosine Monophosphate in Healthy and Crown Gall Tumorous Tissues of Vicia faba

Attempts were made to provide proof for the occurrence of cyclic 3′,5′-adenosine monophosphate in healthy and crown gall tissues of Vicia faba. Although our purified extracts gave positive readings in the Gilman binding assay for cyclic AMP, they were not digested by a specific cyclic 3′,5′-adenosine monophosphate phosphodiesterase from beef heart. The extracts were digested, however, by a partially purified cyclic nucleotide phosphodiesterase from carrot tissue, which attacks both cyclic 2′,3′- and 3′,5′-nucleotides. The data indicate that the substances detected in the V. faba extracts are perhaps cyclic 2′,3′-nucleotides, a possible RNA degradation product.     

Calcium-dependent cyclic nucleotide phosphodiesterase from glial tumor cells

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been identified in homogenates of C-6 glial tumor cells. The Ca²⁺-dependent phosphodiesterase was resolved by ECTEOLA-cellulose chromatography into two fractions. One fraction contained a protein regulator of the enzyme which was identical to a homogeneous Ca²⁺-binding protein (CDR) from porcine brain by the criteria of electrophoretic migration, biological activity, heat stability, and behavior in diverse chromatographic systems. The second fraction contained deactivated enzyme (CDR-dependent phosphodiesterase) which regained full activity upon the readdition of both Ca²⁺ and CDR. In subcellular fractionation experiments both the CDR and the Ca²⁺-dependent phosphodiesterase were predominantly located in the 100,000g supernatant fraction.The apparent K_m values of the phosphodiesterase for cyclic AMP (cAMP) and cyclic GMP (cGMP) were 10 and 1.2 μm, respectively, when CDR was not rate limiting. Minor increases in the apparent K_m for cAMP were observed at rate-limiting concentrations of CDR. At the ratio of CDR to CDR-dependent enzyme present in the C-6 cell homogenate, half-maximal activation was conferred by 4 μm Ca²⁺ for the hydrolysis of 25 μm cGMP and by 8 μm Ca²⁺ for the hydrolysis of 25 μm cAMP. Increased ratios of CDR to CDR-dependent phosphodiesterase increased the sensitivity of the enzyme to Ca²⁺. The enzyme was more sensitive to CDR with cGMP as substrate than with cAMP, and more sensitive at high than at low cyclic nucleotide substrate concentrations. The quantity of enzyme in the assay also influenced the amount of CDR required for half-maximal activation.     

Cyclic adenosine 3',5'-monophosphate phosphodiesterase from Mucor rouxii: regulation of enzyme activity by phosphorylation and dephosphorylation.

Crude preparations of cyclic adenosine 3′, 5′-monophosphate phosphodiesterase were activated 1.5 to 2 fold by incubation with ATP, Mg²⁺ and cyclic AMP in a reaction which was both, time and temperature dependent. Cyclic AMP phosphodiesterase remained in an activated state upon filtration of the enzymatic preparation through Sephadex G-25 and ion-exchange chromatography. Activation of the enzyme in the presence of [γ ³²P]ATP resulted in a significant amount of [³²P] protein-bound radioactivity. Reversible deactivation of cyclic AMP phosphodiesterase was enhanced by Mg²⁺ and was accompanied by the release of [³²P] protein bound radioactivity. The evidence is consistent with a mechanism for controlling cyclic AMP phosphodiesterase through phosphorylation-dephosphorylation sequence.     

An ATP pool with rapid turnover within the cell membrane

Seven-day-old cultures of rat leg muscle cells were double labelled by addition of [¹⁴C] adenine in the culture medium (2 hrs 15 mins) and followed by addition of [³²P] phosphate (15 min). The specific activity (S.A.) of the isolated cyclic [¹⁴C] adenine 3′ – 5′ monophosphate (cAMP) was similar to that of the bulk ATP. The S.A. of [³²P] from cAMP was, however, higher than that of bulk ATP. The S.A. of [³²P] from cAMP could be further modified by prevention of normal muscle cell fusion. It is probable that the cAMP with high [³²P] S.A. was synthesized from a cell membrane pool of ATP with rapid turnover.     

Characterization of the 2',3' cyclic phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase-phosphatase and bacteriophage lambda phosphatase

Clostridium thermocellum polynucleotide kinase-phosphatase (CthPnkp) catalyzes 5′ and 3′ end-healing reactions that prepare broken RNA termini for sealing by RNA ligase. The central phosphatase domain of CthPnkp belongs to the dinuclear metallophosphoesterase superfamily exemplified by bacteriophage λ phosphatase (λ-Pase). CthPnkp is a Ni²⁺/Mn²⁺-dependent phosphodiesterase-monoesterase, active on nucleotide and non-nucleotide substrates, that can be transformed toward narrower metal and substrate specificities via mutations of the active site. Here we characterize the Mn²⁺-dependent 2′,3′ cyclic nucleotide phosphodiesterase activity of CthPnkp, the reaction most relevant to RNA repair pathways. We find that CthPnkp prefers a 2′,3′ cyclic phosphate to a 3′,5′ cyclic phosphate. A single H189D mutation imposes strict specificity for a 2′,3′ cyclic phosphate, which is cleaved to form a single 2′-NMP product. Analysis of the cyclic phosphodiesterase activities of mutated CthPnkp enzymes illuminates the active site and the structural features that affect substrate affinity and k_cat. We also characterize a previously unrecognized phosphodiesterase activity of λ-Pase, which catalyzes hydrolysis of bis-p-nitrophenyl phosphate. λ-Pase also has cyclic phosphodiesterase activity with nucleoside 2′,3′ cyclic phosphates, which it hydrolyzes to yield a mixture of 2′-NMP and 3′-NMP products. We discuss our results in light of available structural and functional data for other phosphodiesterase members of the binuclear metallophosphoesterase family and draw inferences about how differences in active site composition influence catalytic repertoire.     

Preparation and properties of a cell-free, hormonally responsive adenylate cyclase from guinea pig brain

—The accumulation of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) was studied in cell-free homogenates of guinea pig brain. Homogenates, prepared in Krebs-Ringer buffer, responded markedly to the addition of neurohormones with an increased rate of cyclic AMP synthesis; preparations from cerebellum, cerebral cortex, and hippocampus responded to a degree approximating that achieved with slices of these areas of guinea pig brain. Adenylatc cyclase activity was seen only when cyclic AMP was measured by a [³H]adenine prelabelling technique or when total cyclic AMP was measured by radioimmunoassay; [³²P]ATP did not serve as a substrate for this preparation of the enzyme. The adenylate cyclase was paniculate and required a Krebs Ringer buffer; use of tris, or tris with Mg²⁺ and Ca²⁺, resulted in a preparation totally devoid of hormonal stimulation. Digestion by purified beef heart cyclic nucleotide phosphodiesterase, Dowex chromatography, solubility in Ba(OH)₂-ZnSO₄ mixtures, and two thin layer chromatographic systems demonstrated that the product of the hormonally stimulated adenylate cyclase preparation was cyclic AMP. The selectivity of hormonal stimulation and the adrenergic character of the hormonal receptors from different brain areas were maintained in the cell-free preparation. However, simultaneous stimulation with two different neurohormones resulted in additive responses, rather than in the potentiation observed in preparations of slices of brain.     

Uptake and Degradation of Cyclic AMP by Chloronema Cells

Suspension cultures of intact chloronema cells of the moss Funaria hygrometrica take up [³H]cAMP and degrade it rapidly. The increase in total radioactivity accumulated by the cells was linear up to 30 minutes. Initially, the major degradation products were 5′-AMP and adenosine, but later predominantly ADP and ATP. In spite of rapid degradation, the amount of extracellularly applied cAMP retained by the cells is about 4-fold higher than the maximum endogenous level of cAMP reported previously (Handa, Johri 1977 Plant Physiol 59: 490-496). The uptake showed a distinct dependence on the density of the culture. Cells at a lower cell density (1-2 milligrams per milliliter) accumulated 4 to 6 times more radioactivity than the cells at high density (>10 milligrams per milliliter). The cyclic nucleotide phosphodiesterase (cNPDE) activity of whole cells (18 milliunits per milligram protein) was comparable to that of protoplasts (23 milliunits per milligram protein), but about 4-fold lower than that of lysed protoplasts (80 milliunits per milligram protein), indicating an intracellular degradation of cAMP by chloronema cells.     

A batch procedure for the assay of cyclic nucleotide phosphodiesterase.

A procedure for the assay of cyclic nucleotide phosphodiesterase is described in which labeled cyclic nucleotide is separated from labeled nucleoside by the batchwise addition of ethanolic slurries of Dowex 2 fluoride. Under the conditions described there is no detectable adsorption of nucleoside by the anion exchanger, which removes more than 95% of the tritium in boiled samples of [8-³H]cAMP or [8-³H]cGMP. Linear time courses and enzyme vs activity relationships are described for 10^?3 and 10^?7m cAMP and 10^?4m cGMP. The method is limited by interference by neutral salts and by the enzymatic conversion of adenosine into inosine.     

Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain

Membrane-associated, Type II (cGMP-activatable) cyclic nucleotide phosphodiesterase (PDE) from rabbit brain, representing 75% of the total homogenate Type II PDE activity, was purified to apparent homogeneity. The enzyme was released from 13,000 x g particulate fractions by limited proteolysis with trypsin and fractionated using DE-52 anion-exchange, cGMP-Sepharose affinity and hydroxylapatite chromatographies. The enzyme showed 105 kDa subunits by SDS-PAGE and had a Stokes radius of 62.70 A as determined by gel filtration chromatography. Hydrolysis of cAMP or cGMP showed positive cooperativity, with cAMP kinetic behavior linearized in the presence of 2 microM cGMP. Substrate concentrations required for half maximum velocity were 28 microM for cAMP and 16 microM for cGMP. Maximum velocities were approx. 160 mumol/min per mg for both nucleotides. The apparent Kact for cGMP stimulation of cAMP hydrolysis at 5 microM substrate was 0.35 microM and maximal stimulation (3-5-fold) was achieved with 2 microM cGMP. Cyclic nucleotide hydrolysis was not enhanced by calcium/calmodulin. The purified enzyme can be labeled by cAMP-dependent protein kinase as demonstrated by the incorporation of 32P from [gamma-32P]ATP into the 105 kDa enzyme subunit. Initial experiments showed that phosphorylation of the enzyme did not significantly alter enzyme activity measured at 5 microM [3H]cAMP in the absence or presence of 2 microM cGMP or at 40 microM [3H]cGMP. Monoclonal antibodies produced against Type II PDE immunoprecipitate enzyme activity, 105 kDa protein and 32P-labeled enzyme. The 105 kDa protein was also photoaffinity labeled with [32P]cGMP. The purified Type II PDE described here is physicochemically very similar to the isozyme purified from the cytosolic fraction of several bovine tissues with the exception that it is predominantly a particulate enzyme. This difference may reflect an important regulatory mechanism governing the metabolism of cyclic nucleotides in the central nervous system.     

Method for measuring 32P-labeled cAMP levels in intact tissue.

A new method has been developed for prelabeling tissue ATP pools with ³²P inorganic phosphate (P_i) and for the subsequent isolation of [³²P]cAMP and [³²P]ATP. The new method of prelabeling eliminates the need to separate trace amounts of radioactive cAMP from radioactive breakdown products of adenine formed in tissues prelabeled with [³H]- or [¹⁴C]adenine. The effect of epinephrine to increase [³²P]cAMP levels in rat ventral prostate tissue fragments has been studied in terms of increase in the ratio of [³²P]cAMP/[³²P]ATP in the absence and presence of various phosphodiesterase inhibitors. Tissue prelabeling with ³²P_i labels GTP as well as ATP (and other nucleoside triphosphates); thus the method lends itself to the isolation of [³²P]cGMP as well as [³²P]cAMP from the same tissue sample.     

A study of adenosine 3′–5′ cyclic monophosphate binding sites of human erythrocyte membranes using 8-azidoadenosine 3′–5′ cyclic monophosphate,a photoaffinity probe

An earlier report (1a) has shown the utility of 8-N₃cAMP (8-azidoadenosine-3′, 5′-cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that: (1) both cAMP and 8-N₃cAMP stimulate the phosphorylation by [γ-³²P]-ATP of the same red cell membrane proteins; (2) the protein of approximately 48,000 molecular weight whose phosphorylation by [γ-³²P]-ATP is stimulated by cAMP and 8-N₃cAMP migrates at a solwer rate than the protein in the same molecular weight range which is heavily photolabeled with [³²P]-8-N₃cAMP; (3) other cyclic nucleotide binding sites exist besides those initailly reported; (4) the variation in the ratio of incorporation of ³²P-8-N₃cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein.     

A 3′,5′ Cyclic AMP (cAMP) Phosphodiesterase Modulates cAMP Levels and Optimizes Competence in Haemophilus influenzae Rd

Changes in intracellular 3′,5′ cyclic AMP (cAMP) concentration regulate the development of natural competence in Haemophilus influenzae. In Escherichia coli, cAMP levels are modulated by a cAMP phosphodiesterase encoded by the cpdA gene. We have used several approaches to demonstrate that the homologous icc gene of H. influenzae encodes a functional cAMP phosphodiesterase and that this gene limits intracellular cAMP and thereby influences competence and other cAMP-dependent processes. In E. coli, expression of cloned icc reduced both cAMP-dependent sugar fermentation and β-galactosidase expression, as has been shown for cpdA. In H. influenzae, an icc null mutation increased cAMP-dependent sugar fermentation and competence development in strains where these processes are limited by mutations reducing cAMP synthesis. When endogenous production of cAMP was eliminated by a cya mutation, an icc strain was 10,000-fold more sensitive to exogenous cAMP than an icc⁺ strain. The icc strain showed moderately elevated competence under noninducing conditions, as expected, but had subnormal competence increases at onset of stationary phase in rich medium, and on transfer to a nutrient-limited medium, suggesting that excessive cAMP may interfere with induction. Consistent with this finding, a cya strain cultured in 1 mM cAMP failed to develop maximal competence on transfer to inducing conditions. Thus, by limiting cAMP levels, the H. influenzae cAMP phosphodiesterase may coordinate its responses to nutritional stress, ensuring optimal competence development.