Detection of a 3′, 5′-cyclic-AMP phosphodiesterase activity in the lichenized fungus Evernia prunastri

Abstract

A 3′, 5′-cyclic-AMP phosphodiesterase (PDE) was detected and measured in the lichen Evernia prunastri. The percentage of hydrolysis of tritiated 3′, 5′-cyclic-adenosine monophosphate ([³H]-cAMP) and 3′, 5′-cyclic-guanosine monophosphate ([³H]-cGMP) by the PDE enzyme into tritiated 5′-adenosine-monophospahte ([³H]-AMP) and tritiated 5′-guanosine-monophospahte ([³H]-GMP) was measured by treating the PDE products with a 5′-nucleotidase enzyme present in snake venom. The lysate fraction (L) (plasma membranes and cell walls) and the supernatant (S) (soluble fraction of the cells) were tested. In both fractions, competition of unlabelled cAMP, but not unlabelled cGMP, was revealed. Specific competitive PDE inhibitors such as IBMX inhibited enzymatic activity. Although it is thought that in this species cAMP is regulated by red/far red light through PDE activity, this is the first report that seems to suggest the presence of a PDE activity specific for cAMP in lichenized fungi. However, this work is at a preliminary stage and despite the high levels of enzymatic activity with cAMP found in both fractions, data are still insufficient to state the absolute specificity for this nucleotide.     

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.     

A cAMP receptor from mouse liver cytosol whose binding capacity is enhanced by Mg++-ATP.

A receptor with a dissociation constant of 2·10^?6M for cyclic 3′,5′-AMP (cAMP) has been found in mouse liver cytosol. This cAMP binding activity can be differentiated from the cAMP-dependent protein kinase holoenzymes and the free regulatory subunits also found in the cytosol. Mg⁺⁺-ATP increases the number of binding sites for cAMP several fold. This increased capacity for cAMP binding persists after Sephadex G-25 filtration, and incubation for 14 hours in the presence of 5 mM EDTA. Among several adenosine- and guanosine-derivatives tested, only AMP, ADP and ATP compete efficiently with [³H] cAMP for the cAMP binding site.     

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.     

Intracellular signal transduction mechanisms of rat epididymal spermatozoa and their relationship to motility and metabolism

The role of intracellular signal transduction mechanisms in regulating the motility and metabolism of rat spermatozoa in undiluted caudal epididymal fluid (CEF) was examined. Samples of CEF containing immotile spermatozoa were exposed to drugs and other agents that either stimulate signal transduction pathways or mimic the action of their second messengers. Under these conditions, sperm motility in 25–30 nl of CEF was stimulated by calcium ions (Ca²⁺), N,2′ -O-dibutyryl-guanosine 3′:5′ -cyclic monophosphate (dibutryl cGMP), cyclic adenosine 3′:5′-monophosphate (cAMP), N⁶,2′-O-dibutyryladenosine 3′:5′ -cyclic monophosphate (dibutyryl cAMP), 8-bromoadenosine 3′:5′ -cyclic monophosphate (8-bromo cAMP), caffeine, theophylline and bicarbonate ions (HCO₃^?). Other agents such as magnesium ions (Mg²⁺), veratridine, phospholipase C (PLC), ionophore A23187, 1,2-dioctenoyl-sn-glycerol (DAG), phorbol 12-myristate 13-acetate, phospholipase A₂ (PLA₂), arachidonic acid, and melittin did not significantly influence motility. In the presence of radiolabelled energy substrates, untreated (immotile) spermatozoa in samples of CEF utilised D-[U-¹⁴C]glucose and [1-¹⁴C]acetate as exogenous energy sources for oxidative metabolism. No detectable ¹⁴C-lactate was produced, and none of the drugs altered the rate of glycolytic or oxidative metabolism. The findings suggest that the motility of rat caudal epididymal spermatozoa is regulated by Ca²⁺ and the guanylate cyclase and adenylate cyclase pathways, but not through the PLC and PLA₂ pathways. Also, their metabolism of exogenous substrate was uncoupled from the induction of motility, and their oxidative capacity exceeded the rate of flux of glucose-carbon through the glycolytic pathway. © 1994 Wiley-Liss, Inc.     

The relationship of cAMP levels and the rate of pool labelling of cAMP and related bases, nucleosides and nucleotides to the HeLa cell cycle.

Cellular cAMP levels as well as the rate of pool labelling of cAMP and related bases, nucleosides and nucleotides were determined in synchronized cultures of HeLa cells after pulse-labelling with [¹⁴C]adenine. The cAMP levels were found to be maximal in G 1 and minimal in G 2 and mitosis, as previously reported by others. The rate of labelling of the cAMP pools, however, was found to be maximal in G 2 and decreased to a minimum in G 1. This suggests that the rate of cAMP synthesis is highest when pool level is lowest and vice versa. A comparison of cAMP levels and the rate of 5′AMP pool labelling throughout the HeLa cell cycle indicated an inverse relationship. Such a relationship emphasizes the role of the cyclic 3′,5′-phosphodiesterase activity during the cell cycle. The kinetics of pool labelling of IMP, ATP, and hypoxanthine throughout the cell cycle suggested that the adenylate energy charge fluctuated as a function of the cell cycle. The apparent activation of the adenylate cyclase during G 2 and mitosis as reflected by the increased rate of cAMP pool labelling suggests that the super phosphorylation of H 1 histone during G 2-mitotic transition may be mediated by cAMP-dependent phosphokinases.     

Increase of cyclic AMP-dependent protein kinase type II as an early event in hormone-dependent mammary tumor regression.

Primary, 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary carcinoma in the rat contains cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent and -independent forms of protein kinase. When growth of DMBA-induced tumors was arrested by either ovariectomy or N⁶,O²′-dibutyryl cAMP treatment of the host, the activity of cAMP-dependent protein kinase type II markedly increased in the tumor cytosol, as shown by DEAE-cellulose chromatography and autophosphorylation. The increase in activity of cAMP-dependent protein kinase was also demonstrable in the tumor cytosol and nuclei following $\text{in}$$\text{vitro}$ incubation of tumor slices with cAMP. These results suggest that protein kinase type II is involved in the regression of hormone-dependent mammary tumors.     

Modulation by gibberellic acid and adenosine-3′,5′-cyclic monophosphate of starch hydrolysing activity of cowpea seedlings

In cowpea seedlings starch hydrolysing activity increases 35–50 fold on germination for 4 days. This increase in enzyme activity was inhibited by the in vivo addition of 1% glucose but this inhibition was completely overcome by the addition of gibberellic acid (GA₃) (10^?5 M) and adenosine-3′,5′-cyclic monophosphate (cAMP) (10^?5 M). At 5% glucose, GA₃ and cAMP were only partially effective. Structural analogues of cAMP failed to relieve the inhibitory effect of glucose. The inhibition by glucose is not direct but RNA and protein synthesis may be involved. Glucose appears to reduce the internal pool of cAMP which causes inhibition of RNA synthesis and decrease in starch hydrolysing activity. Exogenous application of cAMP may replenish the endogenous pool of cyclic nucleotide and thus overcome inhibition of RNA synthesis and enzyme activity.     

Total polar lipid biosynthesis during growth of Crotalaria juncea pollen tubes

[1-¹⁴C]-Acetate incorporation into total and polar lipids was studied in the growing pollen tubes of Crotalaria juncea. Ungerminated pollen had phosphatidyl inositol, phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, monogalactosyl diglyceride, digalactosyl diglyceride, sulpholipid and steryl glycosides. In the growing pollen tubes considerable [1-¹⁴C]-acetate incorporation was observed into the individual polar lipids. The exogenous carbon source significantly influenced lipid biosynthesis. Boric acid (20mg/l.) promoted both pollen tube growth and acetate incorporation into phospholipids. In comparison to 5′-adenosine monophosphate, cyclic-3′,5′-adenosine monophosphate (cAMP) promoted tube growth and also enhanced phospho-and glycolipid biosynthesis. The regulation of membrane component biosynthesis by cAMP is suggested.     

Bursicon-induced adenyl cyclase activity in the haemocytes of the American cockroach

Partially purified preparations of bursicon were injected, between ecdyses, into American cockroaches (Periplaneta americana) held at 4°C. After short periods of time, the haemocytes were removed and found to contain appreciable amounts of cyclic adenosine 5′-monophosphate (cAMP). Identical experiments using animals reared at 23°C did not result in the accumulation of cAMP. These and additional data suggest that phosphodiesterase is not active during the initial stages of bursicon-stimulated adenyl cyclase.Incubation of haemocyte soluble proteins in the presence of [³H]cAMP, followed by molecular sieve chromatography on P-60 polyacrylamide gel, shows the binding of cAMP to large molecular weight proteins. Similar experiments, but with phosphodiesterase inhibition revealed a reduced level of radioactive bound protein. From these studies one can assume that the haemocytes contain a soluble cAMP-dependent protein kinase.If either [¹⁴C]-leucine or [¹⁴C]-tyrosine is incubated with whole blood taken from animals between ecdyses, little or no uptake by the haemocytes can be seen. Identical studies, but with the addition of cAMP, results in a dramatic enhancement of amino acid uptake. It appears that bursicon stimulates blood-cell adenyl cyclase, which in turn produces cAMP. The cAMP subsequently activates a specific protein kinase that phosphorylates potential membrane proteins. The membrane phosphoprotein may be instrumental in the enhanced uptake of amino acids.     

5′-Nucleotidase: Solubilization,radiochemical analysis,and electrophoresis

5′-Nucleotidase (5′-NT, E.C. 3.1.3.5) of cultured human and rodent cells was rendered soluble using the zwitterionic detergent Zwittergent 314. Optimal activity of 5′-NT was obtained when sonicated cells were incubated in solutions containing 0.75% (w/v) Zwittergent. A method was developed for the determination of the activity of 5′-NT in which the unutilized substrate, [¹⁴C]-AMP. was precipitated with lanthanum chloride and the soluble [¹⁴C]-adenosine was measured by scintillation counting. 5′-NT isozymes were separated using agarose gel electrophoresis and isoelectric focusing in polyacrylamide gel. The zones of enzyme activity were established by precipitation of unutilized [¹⁴C]-AMP with LaCl₃, removal of soluble [¹⁴C]-adenosine by washing gels in water, and autoradiography. The zones of 5′-NT appeared as clear zones on darkened X-ray film. When analyzed by agarose gel electrophoresis, fibroblasts derived from human skin and rat liver produced a single zone of 5′-NT activity. The 5′-NT isozyme of rat cells migrated faster than that of human cells and was easy to distinguish. The presence of detergent in the sample and in the gel enhanced enzymatic activity and improved the separation of the isozymes. Isoelectric focusing resolved 5′-NT of human fibroblasts into two molecular forms. one of which focused in the region of pH 6 and the other at pH 5.     

DYNAMICS OF CELLULAR AND EXTRACELLULAR cAMP IN ANABAENA FLOS-AQUAE (CYANOPHYTA): INTRINSIC CULTURE VARIABILITY AND CORRELATION WITH METABOLIC VARIABLES1

The production and extracellular release of cyclic adenosine 3′: 5′-monophosphate (cAMP) by the blue-green alga Anabaena flos-aquae (Lyngb.) Breb. varied greatly within and between active growth phase and stationary phase and under differing nutrient regimes. Enhanced cellular cAMP production was found in actively growing Anabaena inoculated into media deficient in nitrate or phosphate, or into fresh media containing non-limiting nutrient concentrations. In stationary phase Anabaena, but not actively growing cells, the concentrations of intra-cellular cAMP present in cells grown under a variety of nutrient regimes could be significantly correlated to [¹⁴C]-bicarbonate uptake by an exponential relationship.     

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.     

Preparative solvent partition chromatography of butyrylated cyclic AMP analogues

A simple and effective separation of cyclic adenosine-3′,5′-monophosphate (cAMP) and its butyryl-substituted analogues using partition chromatography on columns of Sephadex gel in isopropanol/0.5 m ammonium acetate (4:1) is described. The technique is suitable for preparative separations as demonstrated by revised uv spectral data obtained on butyrylated cAMP's purified by this technique. In addition, it has analytical utility in that it allows complete separation of N⁶-monobutyryl cAMP from O^2′-monobutyryl cAMP, thereby permitting simultaneous and independent assessment of the rate of acyl substituent hydrolysis from the disubstituted derivative (N⁶,O^2′-dibutyryl cAMP), and this is demonstrated under several conditions.     

A simple radioimmunoassay for 3',5', cyclic adenosine monophosphate.

A radioimmunoassay for 3′,5′ cAMP has been developed in which [³H]3′,5′ cAMP is the radioligand. Antibody-bound and free fractions are separated with dextran-coated charcoal. The sensitivity of the assay is 0.03 pmoles and antiserum specificity is 7 orders with respect to other adenine nucleotides. Samples are prepared by ethanol precipitation. Tissue levels of 3′,5′ cAMP are comparable to those reported by others.     

Effect of cAMP on nucleoside metabolism. I. Effect on thymidine transport and incorporation in monkey cells (CV-1)

The effect of cAMP on a monkey kidney cell line (CV-1) noninfected and infected by SV40 was studied. No effect was found on either growth rate or cell morphology when concentrations up to 1 mM of 3′5′ cAMP were used. However, cAMP was found to increase the incorporation of ³H-thymidine into both cellular and viral DNA without a net increase in DNA synthesis. This increased incorporation was found to be related to an enhanced uptake of thymidine into the nucleotide pool which is reflected in an increase of phosphorylated nucleotides. This, coupled with a lack of effect of cAMP on endogenous deoxyribonucleotide production, produces an increased specific activity of the deoxyribonucleotide triphosphates, with a resultant increase in specific activity of DNA.     

Phosphodiesterase induction in Dictyostelium discoideum by inhibition of extracellular phosphodiesterase activity

Adenosine 3′,5′-monophosphate (cAMP) is a chemoattractant in Dictyostelium discoideum; it also induces phosphodiesterase activity. Recently it was shown (M. H. Juliani, J. Brusca, and C. Klein, (1981)Develop. Biol.83, 114–121) that N⁶-(aminohexyl)adenosine 3′,5′-monophosphate (hexyl-cAMP) effectively induced phosphodiesterase activity, while this compound was chemotactically inactive and did not effectively bind to the cell surface receptor for cAMP. It was suggested that hexyl-cAMP and cAMP induce phosphodiesterase activity via a chemoreceptor-independent mechanism. In another recent report (P. J. M. Van Haastert, R. C. Van der Meer, and T. M. Konijn (1981)J. Bacteriol.147, 170–175) investigation of induction of phosphodiesterase by several cAMP derivatives revealed that phosphodiesterase induction and chemotaxis had similar cyclic nucleotide specificity. Based on this result it was suggested that cAMP induces phosphodiesterase activity via activation of the chemotactic receptor. In this report we show that hexyl-cAMP transiently inhibits extracellular and cell surface phosphodiesterase. This transient inhibition of the inactivating enzyme and the permanent release of small amounts of cAMP by the cells leads to a transient increase of extracellular cAMP levels. Hexyl-cAMP does not inhibit beef heart phosphodiesterase, and is not degraded by this enzyme. Addition of hexyl-cAMP to a cell suspension containing beef heart phosphodiesterase does not result in an accumulation of extracellular cAMP, and phosphodiesterase induction is absent. We conclude that hexyl-cAMP inhibits phosphodiesterase activity which leads to the accumulation of cAMP; consequently cAMP binds to the chemotactic cAMP receptor resulting in the induction of phosphodiesterase activity.     

Effects of the cilioexcitatory neurohumors dopamine and 5-hydroxytryptamine on cyclic AMP levels in the gill of the mussel Mytilus edulis.

Cyclic 3′, 5′-adenosine monophosphate (cAMP) has been identified in the ciliated gill epithelium of the marine mussel $\text{Mytilus}$$\text{edulis}$. In concentrations which stimulate the rate of particle transport by frontal gill cilia, DA and 5HT stimulate levels of cAMP within the gill. The stimulation occurs in as early as 15 sec and is graded from 10^?6M to 10^?4M. DA plus 5HT is not additive at maximal effective concentrations of both amines. ACH does not mimic the DA or 5HT stimulation of cAMP. Theophylline alone has a weak effect on cAMP levels; however, the effect of theophylline is potentiated in the presence of DA or 5HT. Dibutyryl cAMP produces a gradual stimulation in the rate of particle transport. It is suggested that the dopaminergic and serotonergic excitatory control of particle transport by frontal gill cilia of $\text{Mytilus}$$\text{edulis}$ is mediated through a cAMP second messenger system.     

Effects of lipids on cyclic-nucleotide phosphodiesterases

Several naturally-occurring lipids but not n-propanol, guanidine-HCl or a variety of synthetic detergents stimulate the 3′,5′-cyclic AMP-phosphodiesterase activities of a supernatant fraction of brain at 1.25 × 10^?7 M cAMP. The time courses of the reaction are linear in the presence and absence of lipid. On the other hand, lipid has different effects on various phosphodiesterase activities in fractions obtained after gel filtration of the crude extract. It stimulates the phosphodiesterase activities measured at 1.25 × 10^?7 M and 10^?4 M 3′,5′-cyclic-AMP and 1.25 × 10^?7 M 3′,5′-cyclic GMP in two of the fractions partially retained in the gel. However, lipid has little effect on the enzymatic hydrolysis of low concentrations of cAMP or cGMP and markedly inhibits the hydrolysis of high concentrations of cAMP by the fraction excluded from the gel.