Effects of zinc chloride on the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart.

In the presence of 10 micrometer Ca2+ and 5 mM Mg2+ (or 0.25 mM Mg2+), the addition of 100 micrometer Zn2+, Ni2+, Co2+, Fe2+, Cu2+ or 1 mM Mn2+ resulted in varying degrees of stimulation or inhibition of 10(-6) M cyclic GMP and cyclic AMP hydrolysis by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart in the absence or presence of phosphodiesterase activator. The substrate specificity of the enzyme was altered under several conditions. The addition of Zn2+ in the presence of 5 mM Mg2+ and the absence of activator resulted in the stimulation of cyclic GMP hydrolysis over a narrow substrate range while reducing the V 65% due to a shift in the kinetics from non-linear with Mg2+ alone to linear in the presence of Zn2+ and Mg2+. Zn2+ inhibited the hydrolysis of cyclic GMP and cyclic AMP in the presence of activator with Ki values of 70 and 100 micrometer, respectively. Zn2+ inhibition was non-competitive with substrate, activator and Ca2+ but was competitive with Mg2+. In the presence of 10 micrometer Ca2+ and activator, a Ki of 15 micrometer for Zn2+ vs. Mg2+ was noted in the hydrolysis of 10(-6) M cyclic GMP. Several effects of Zn2+ are discussed which have been noted in other studies and might be due in part to changes in cyclic nucleotide levels following phosphodiesterase inhibition.     

Effects of CuCl2 on the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart

CuCl₂ non-comepetitively inhibited the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent phosphodiesterase from bovine heart in the presence of 5 mM Mg²⁺, 10 μM Ca²⁺ and phosphodiesterase activator with K_i values of approximately 2 μM for both substrates. CuCl₂ inhibition was also non-competitive with Mg²⁺, Ca²⁺ and phosphodiesterase activator. Dialysis demonstrated that CuCl₂ inhibition in reversible. Treatment of the enzyme with p-hydroxymercuribenzoate resulted in the loss of enzyme activity, suggesting the presence of sulfhydryl groups essential for enzyme activity. The inhibitory activity of CuCl₂ was not additive with that p-hydroxymercuribenzoate, therefore CuCl₂ may inhibit enzyme activity by binding to one or more essential sulfhydryl groups. CuCl₂ also inhibited the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase from bovine heart with an I₅₀ value of 18 μM. Several effects of Cu²⁺ are discussed which have been noted in other studies and might be due, in part, to changes in cyclic nucleotide levels following alterations in phosphodiesterase activity.     

Cyclic 3',5'-AMP phosphodiesterase of rabbit aorta.

Cyclic AMP and cyclic GMP phosphodiesterase activities (3' : 5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca2+ in the presence of the activator. However, the activity of the cyclic AMP phosphodiesterase was not significantly stimulated by Ca2+ up to 100 muM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60-70%) of the media enzyme was found in 105 000 times g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 times g supernatant the main kinetic parameters were specified as follows: 1) The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg2+ was achieved at 4mM of MgC12. 2) High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3) Activity-substrate concentration relationship revealed a high affinity (Km equals 1.0 muM) and low affinity (Km equals 45 muM) for cyclic AMP. The homogenate and 105 000 times g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent Km values for cyclic AMP hydrolysis were 1.2 muM and 36-40 muM and an enzyme extracted by sonication from 105 000 times g precipitate also exhibited non-linear kinetics (Km equals 5.1 muM and 70 muM). 4) Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I5 o at 0.62 muM, I5 o of the media at 0.62 muM and I5 o of the adventitia at 1.0 muM) than on the brain (I5 o at 8.5 muM) and serum (I5 o at 20 muM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.     

Multiple cyclic nucleotide phosphodiesterase activities from rat tissues and occurrence of a calcium-plus-magnesium-ion-dependent phosphodiesterase and its protein activator.

1. Supernatant fluids from rat cerebral cortex, cerebellum, kidney, heart and liver contained more phosphodiesterase activity hydrolysing cyclic GMP than that hydrolysing cyclic AMP when assayed with sub-saturating concentrations of substrate. 2. These activities were resolved into several fractions by Sephadex G-200 gel filtration; no two tissues had similar activity profiles. 3. With every tissue examined, a fraction (fraction II) with a molecular weight of about 150,000 was obtained which hydrolysed cyclic GMP preferentially at sub-saturating substrate concentrations in the presence of micromolar concentration of Ca2+, millimolar concentration of Mg2+ and a protein activator. 4. The activity of fraction II accounted for about 60 percent in liver, more than 80 percent in heart and cerebellum, and almost 100 percent in cerebral cortex of the total activity for cyclic GMP hydrolysis, calculated from the activity profiles. 5. Km values of fraction II samples from kidney, heart and liver for cyclic GMP were 1.3, 1.7 and 5 muM respectively. 6. 3-Isobutyl-1-methylxanthine inhibited hydrolysis of cyclic GMP by fraction II with an I50 value of 3muM for heart and liver and 50 muM for cerebrum. 7. The activator protein, with an estimated molecular weight of about 30,000 was isolated from all the tissues listed in 1.8. The concentrations of activator protein and of the isolated enzyme, fraction II, did not correspond exactly.     

Catalytic and regulatory properties of two forms of bovine heart cyclic nucleotide phosphodiesterase.

The soluble supernatant fraction of bovine heart homogenates may be fractionated on a DEAE cellulose column into two cyclic nucleotide phosphodiesterases (EC 3.1.4.-):PI and PII phosphodiesterases, in the order of emergence from the column. In the presence of free Ca2+, the PI enzyme may be activated several fold by the protein activator which was discovered by Cheung((1971) J. Biol. Chem. 246, 2859-2869). The PII enzyme is refractory to this activator, and is not inhibited by the Ca2+ chelating agent, ethylene glycol bis (beta-aminoethyl ether)-N, N'-tetraacetate (EGTA). The activated activity of PI phosphodiesterase may be further stimulated by imidazole or NH+4, and inhibited by high concentrations of Mg2+. These reagents have no significant effect on either the PII enzyme or the basal activity of PI phosphodiesterase. Although both forms of phosphodiesterase can hydrolyze either cyclic AMP or cyclic GMP, they exhibit different relative affinities towards these two cyclic nucleotides. The PI enzyme appears to have much higher affinities toward cyclic GMP than cyclic AMP. Km values for cyclic AMP and cyclic GMP are respectively 1.7 and 0.33 mM for the non-activated PI phosphodiesterase; and 0.2 and 0.007 mM for the activated enzyme. Each cyclic nucleotide acts as a competitive inhibitor for the other with Ki values similar to the respective Km values. In contrast with PI phosphodiesterase, PII phosphodiesterase exhibits similar affinity toward cyclic AMP and cyclic GMP. The apparent Km values of cyclic AMP and cyclic GMP for the PII enzyme are approx. 0.05 and 0.03 mM, respectively. The kinetic plot with respect to cyclic GMP shows positive cooperativity. Each cyclic nucleotide acts as a non-competitive inhibitor for the other nucleotide. These kinetic properties of PI and PII phosphodiesterase of bovine heart are very similar to those of rat liver cyclic GMP and high Km cyclic AMP phosphodiesterases, respectively (Russel, Terasaki and Appleman, (1973) J. Biol. Chem. 248, 1334).     

Cyclic nucleotide phosphodiesterases from rat anterior pituitary. Characterization of multiple forms and regulation by protein activator and Ca+.

Phosphodiesterase activities for adenosine and guanosine 3':5'-monophosphates (cyclic AMP and cyclic GMP) were demonstrated in particulate and soluble fractions of rat anterior pituitary gland. Both fractions contained higher activity for cyclic GMP hydrolysis than that for cyclic AMP hydrolysis when these activities were assayed at subsaturating substrate concentrations. Addition of protein activator and CaCl2 to either whole homogenate, particulate or supernatant fraction stimulated both cyclic AMP and cyclic GMP phosphadiesterase activities. Almost 80% of cyclic AMP and 90% of cyclic GMP hydrolyzing activities were localized in soluble fraction. Particulate-bound cyclic nucleotide phosphodiesterase activity was completely solubilized with 1% Triton X-100. Detergent-dispersed particulate and soluble enzymes were compared with respect to Ca2+ and activator requirements and gel filtration profiles. Particulate, soluble and partially purified phosphodiesterase activities were also characterized in relation to divalent cation requirements, kinetic behavior and effects of Ca2+, activator and ethyleneglycol-bis-(2-aminoethyl)-N,N'-tetraacetic acid. Gel filtration of either sonicated whole homogenate or the 10500 X g supernatant fraction showed a single peak of activity, which hydrolyzed both cyclic AMP and cyclic GMP and was dependent upon Ca2+ and activator for maximum activity. Partially purified enzyme was inhibited by 1-methyl-3-isobutylxanthine and papaverine with the concentration of inhibitor giving 50% inhibition at 0.4 muM substrate being 20 muM and 24 muM for cyclic AMP and 7 muM and 10 muM for cyclic GMP, respectively. Theophylline, caffeine and theobromine were less effective. The rat anterior pituitary also contained a protein activator which stimulated both pituitary cyclic nucleotide phosphodiesterase(s) as well as activator-deficient brain cyclic GMP and cyclic AMP phosphodiesterases. Chromatography of the sonicated pituitary extract on DEAE-cellulose column chromatography resolved the phosphodiesterase into two fractions. Both enzyme fractions hydrolyzed cyclic AMP and cyclic GMP and had comparable apparent Km values for the two nucleotides. Hydrolysis of cyclic GMP and cyclic AMP by fraction II enzyme was stimulated 6--7-fold by both pituitary and brain activator in the presence of micromolar concentrations of Ca2+.     

Effects of divalent metals on the specificity of inhibitors of the cyclic nucleotide phosphodiesterases from bovine heart

Divalent metals used to support phosphodiesterase (EC 3.1.4.-) activity have been found to influence the substrate and enzyme specificity of many phosphodiesterase inhibitors in studies of the hydrolysis of cyclic AMP and cyclic GMP by the calmodulin-dependent and cyclic AMP-specific phosphodiesterases from bovine heart. Many compounds displayed marked differences in substrate specificity and inhibitory potency in the presence of Mg2+, as compared with Mn2+, when studied with the unactivated form of calmodulin-dependent phosphodiesterase, while few compounds displayed differences in the presence of calmodulin. With a single divalent metal, marked differences in inhibitory potency and substrate specificity were also observed in the absence or presence of calmodulin suggesting that alterations in calmodulin and/or Ca2+ levels may greatly affect the response to phosphodiesterase inhibitors. Divalent metals did not alter the effects of inhibitors on the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase, however divalent metals would probably indirectly influence the relative cellular level of cyclic AMP hydrolyzed by this enzyme, and therefore the effects of inhibitors, through metal effects on the calmodulin-dependent phosphodiesterase. No correlation was found between the inhibitory activity of the compounds, many of which were cyclic nucleotide analogs, and their ability to activate cyclic AMP-dependent or cyclic GMP-dependent protein kinases or to affect cyclic AMP-dependent protein kinase activity by displacing bound cyclic AMP.     

A kinetic analysis of the cyclic nucleotide phosphodiesterase regulation by the endogenous protein activator. A study of rat brain and frog sympathetic chain.

The effect of the endogenous protein activator on the kinetic characteristics of a highly purified, activator-deficient rat brain phosphodiesterase (EC 3.1.4.-) of a highly purified, activator-deficient rat brain phosphodiesterase (EC 3.1.4-) was studied. This enzyme preparation has only a high Km for cyclic AMP and a low Km for cyclic GMP. In the presence of 20 muM Ca2+, saturating concentrations of the activator decreased the Km of this enzyme for cyclic AMP from 350 muM to about 80 muM, without changing the V. The phosphodiesterase activator did not change the Km of phosphodiesterase for cyclic GMP; however, amoderate increase of V was seen. The activator lacks species specificity; the activator isolated from the bullfrog sympathetic chain produced the same qualitative and comparable quantitative changes in the kinetic properties of the purified rat brain phosphodiesterase. Cyclic GMP is a potent competitive inhibitor of the phosphodiesterase activation by the activator (Ki=1.8 muM), using cyclic AMP as a substrate. Cyclic AMP inhibits slightly the hydrolysis of cyclic GMP by phosphodiesterase in the presence of activator (Ki=155 muM) only.     

The effect of Ca++ on cyclic nucleotide phosphodiesterases of superior cervical ganglion.

Cyclic nucleotide phosphodiesterase was examined in canine and bovine superior cervical ganglia. Activity in crude supernatant fractions was only slightly stimulated by Ca++ despite the presence of protein activating factor. Three forms of phosphodiesterase were resolved from bovine ganglia supernatant extracts by chromatography on DEAE-cellulose. The first enzyme eluted, (DI), was almost completely specific for cyclic GMP, while the other two (DII and DIII), hydrolyzed both cyclic AMP and cyclic GMP; all were free of heat-stable protein activator. Each enzyme was inhibited by low concentrations of Ca++ in the assay medium. Inhibition by Ca++ was reversed by addition of protein activator, but activity did not increase above the control level. Cyclic AMP hydrolysis by enzyme DII was stimulated by micromolar concentrations of cyclic GMP. This stimulation was reduced by Ca++ unless protein activator was present.     

Cyclic nucleotide phosphodiesterase activities of pig coronary arteries.

Most (85% or more) of the cyclic nucleotide phosphodiesterase (3' :5' -cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) activity of pig coronary arteries was found in the 40 000 times g supernatant fraction of homogenates of the intima plus media layer. Chromatography of the soluble fraction of this layer on DEAE-cellulose resolved two phosphodiesterase activities and a heat stable, non-dializable activator. Peak I activity had apparent Km values of 2-4 muM for cyclic GMP and 40-100 muM for cyclic AMP. Peak II activity was relatively specific for cyclic AMP and exhibited apparent negatively cooperative behavior. Peak I but not peak II activity could be stimulated 3-8-fold by the addition of the boiled activator fraction or a boiled crude supernatant fraction. Cyclic AMP hydrolysis by peak I or peak II was more rapid in the presence of Mn-2+ than Mg-2+, but the latter promoted hydrolysis of cyclic GMP by peak I more effectively than did Mn-2+ in the presence of activator. In the absence of added metals, ethylene bis(oxyethylenenitriol)tetra-acetic acid (EGTA) and EDTA both inhibited hydrolysis of cyclic AMP and cyclic GMP by phosphodiesterase activities in the supernatant fraction and in peak I, but EDTA produced more complete inhibition at lower concentrations than did EGTA. Imidazole (1 muM to 10 mM) had virtually no effect on the hydrolysis of cyclic AMP or cyclic GMP catalyzed by either of the two separated peaks or by total phosphodiesterase activities in crude supernatant or particulate fractions.     

Protein activator of cyclic AMP phosphodiesterase and cyclic nucleotide phosphodiesterase in bovine retina and bovine lens. Activity, subcellular distribution and kinetic parameters.

We have examined the activity of cyclic AMP phosphodiesterase, cyclic GMP phosphodiesterase and the protein activator of cyclic AMP phosphodiesterase in various anatomic and subcellular fractions of the bovine eye. Cyclic GMP hydrolysis was 1.6--12 times faster than hydrolysis of cyclic AMP in the subcellular fractions of the retina and in the precipitate of the rod outer segment. An opposite pattern was seen in the bovine lens, where the hyrolysis of cyclic AMP occurred 17 and 169 times faster than that of cyclic GMP in the supernatant and precipitate of lens, respectively. The activity of cyclic AMP phosphodiesterase was not affected by ethylene-glycol bis(beta-aminoethylether)-N,N'-tetraacetic acid in any fractions except in the retinal supernatant, suggesting that the phosphodiesterase exists primarily as a Ca2+-independent, activator-independent form. However, the protein activator of cyclic AMP phosphodiesterase existed in all fractions examine. A complex kinetic patternwas observed for both cyclic AMP and cyllic GMP hydrolysis by the 105000 times g lens supernatant. The Michaelis constants for both cyclic AMP (1.3-10(-6) and 9.I-10(-6) M) and cyclic GMP (1.04-10(6) AND 1.22 10(-5) M) appeared to be similar.     

Activity of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by bovine heart and rat liver cyclic nucleotide phosphodiesterases.

The effects of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by crude and partially purified phosphodiesterases obtained from bovine heart and rat liver were studied in order to determine if imidazole has an activity on cyclic nucleotide hydrolysis under conditions which might explain its ability to antagonize the effects of several hormones. Imidazole-Cl (40 mm, pH 7.4) had no effect on the hydrolysis of cyclic AMP or cyclic GMP at substrate levels below 10 μm by the crude enzymes but increasing stimulation was observed with increasing substrate concentrations reaching a twofold stimulation at 1 mm cyclic nucleotide. Three phosphodiesterases with varying substrate specificities were partially purified from bovine heart by ammonium sulfate precipitation and diethyl aminoethyl cellulose chromatography. With these enzymes imidazole had less stimulatory activity and some inhibitory effect on the hydrolysis of 10^?4m cyclic AMP and cyclic GMP but was without significant effect on the hydrolysis of 10^?6m cyclic AMP or cyclic GMP. The stimulatory activity of imidazole on the hydrolysis of high levels of cyclic nucleotide was dependent on the presence of phosphodiesterase activator. The stimulatory effect of the activator and imidazole plus activator on the hydrolysis of 10^?4m cyclic GMP by the rather cyclic GMP-specific enzyme could be eliminated by the addition of ethylene glycol-bis-(β-aminoethyl ether)N,N′-tetraacetate (EGTA) and restored by Ca²⁺. Imidazole was without effect on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase from bovine heart. The lack of effect of imidazole on the hydrolysis of physiological levels of cyclic AMP or cyclic GMP suggests that the activity of imidazole to antagonize the effects of various hormones is probably not due to a direct action of imidazole on the hydrolysis of cyclic AMP or cyclic GMP.     

Studies on cyclic nucleotide metabolism in Tetrahymena pyriformis: partial characterization of cyclic AMP- and cyclic GMP-dependent phosphodiesterases

Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction; the activities were optimal at pH 8.0-9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg2+ or Mn2+. A kinetic analysis of the properties of the enzymes yielded 2 apparent K(m) values ranging in concentration from 0.5 to 50 micron and from 0.1 to 62 micron for cyclic AMP and GMP, respectively. A Ca2+ -dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca2+, although this activator did not stimulate the phosphodiesterase. The results suggested that Tetrahymena might contain 2 types of Ca2+ -dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.     

Ca2+-independent cyclic GMP phosphodiesterases from rat liver and HTC hepatoma cells.

We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.     

The effect of intracellular calcium ions on adrenaline-stimulated adenosine 3'':5''-cyclic monophosphate concentrations in pigeon erythrocytes, studied by using the ionophore A23187.

1. The bivalent cation ionophore A23187 was used to increase the intracellular concentration of Ca2+ in pigeon erythrocytes to investigate whether the increase in cyclic AMP content caused by adrenaline might be influenced by a change in intracellular Ca2+ in intact cells. 2. Incubation of cells with adrenaline, in the concentration range 0.55--55 muM, resulted in an increase in the concentration of cyclic AMP over a period of 60 min. The effect of adrenaline was inhibited by more than 90% with ionophore A23187 (1.9 muM) in the presence of 1 mM-Ca2+. This inhibition could be decreased by decreasing either the concentration of the ionophore or the concentration of extracellular Ca2+, and was independent of the concentration of adrenaline. 3. The effect of ionophore A23187 depended on the time of incubation. Time-course studies showed that maximum inhibition by ionophore A23187 was only observed when the cells were incubated with the ionophore for at least 15 min before the addition of adrenaline. 4. The inhibition by ionophore A23187 depended on the concentration of extracellular Ca2+. In the absence of Mg2+, ionophore A23187 (1.9 muM) inhibited the effect of adrenaline by approx. 30% without added Ca2+, by approx. 66% with 10 muM-Ca2+ and by more than 90% with concentrations of added Ca2+ greater than 30 muM. However, even in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate](0.1--10 mM), ionophore A23187 caused an inhibition of the cyclic AMP response of at least 30%, which may have been due to a decrease in cell Mg2+ concentration. 5. The addition of EGTA after incubation of cells with ionophore A23187 resulted in a partial reversal of the inhibition of the effect of adrenaline. 6. Inclusion of Mg2+ (2 mM) in the incubation medium antagonized the inhibitory action of ionophore A23187. This effect was most marked when the ionophore A23187 was added to medium containing Mg2+ before the addition of the cells. 7. The cellular content of Mg2+ was decreased by approx. 50% after 20 min incubation with ionophore A23187 (1.9 muM) in the presence of Ca2+ (1 mM) but no Mg2+. When Mg2+ (2 mM) was also present in the medium, ionophore A23187 caused an increase of approx. 80% in cell Mg2+ content. Ionophore A23187 had no significant effect on cell K+ content. 8. Ionophore A23187 caused a decrease in cell ATP content under some conditions. Since effects on cyclic AMP content could also be shown when ATP was not significanlty lowered, it appeared that a decrease in ATP in the cells could not explain the effect of ionophore A23187 on cyclic AMP. 9. Ionophore A23187 (1.9 muM), with 1 mM-Ca2+, did not enhance cyclic AMP degradation in intact cells, suggesting that the effect of ionophore A23187 on cyclic AMP content was mediated through an inhibition of adenylate cyclase rather than a stimulation of cyclic AMP phosphodiesterase. 10. It was concluded that in intact pigeon erythrocytes adenylate cyclase may be inhibited by intracellular concentrations of Ca2+ in the range 1-10 muM.     

A stereochemical investigation of the hydrolysis of cyclic AMP and the (Sp)-and (Rp)-diastereoisomers of adenosine cyclic 3'':5''-phosphorothioate by bovine heart and baker''s-yeast cyclic AMP phosphodiesterases.

Bovine heart cyclic AMP phosphodiesterase, which has a requirement for Mg2+, hydrolyses cyclic AMP with inversion of configuration at the phosphorus atom, but only the (Sp)-diastereoisomer of adenosine cyclic 3':5'-phosphorothioate is hydrolysed by this enzyme. By contrast, the low-affinity yeast cyclic AMP phosphodiesterase, which contains tightly bound Zn2+, hydrolyses both the (Sp)- and the (Rp)-diastereoisomers of adenosine cyclic 3':5'-phosphorothioate, the (Rp)-diastereoisomer being the preferred substrate under V max. conditions. Both of the diastereoisomers of adenosine cyclic 3':5'-phosphorothioate, as well as cyclic AMP, are hydrolysed with inversion of configuration at the phosphorus atom by the yeast enzyme. It is proposed that, with both enzymes, the bivalent metal ion co-ordinates with the phosphate residue of the substrate, and that hydrolysis is catalysed by a direct "in-line' mechanism.     

Characterization of a calmodulin-dependent high-affinity cyclic AMP and cyclic GMP phosphodiesterase from male mouse germ cells.

Two cyclic nucleotide phosphodiesterase activities were separated by ion-exchange chromatography of cytosol from male mouse germ cells. A form eluted at low salt concentration showed high affinity (Km congruent to 2 microM) and low affinity (Km congruent to 20 microM) for cyclic AMP, and high affinity (Km congruent to 3.5 microM) for cyclic GMP. A second form, eluted at high salt concentration, showed high affinity (Km congruent to 5 microM) for cyclic AMP and was similar to a phosphodiesterase activity described in rat germ cells. The present study was performed to characterize the first form, which represents most of the phosphodiesterase activity in mouse germ cells. The enzyme was sensitive to Ca2+ and calmodulin stimulation, which increased its activity 3-4-fold. Calmodulin stimulation depended on direct interaction of the activator with the enzyme, as indicated by the reversible changes in the chromatographic elution pattern in the presence of Ca2+, as well as by the increase in the sedimentation coefficient in the presence of calmodulin. Reciprocal inhibition kinetics between cyclic AMP and cyclic GMP for the calmodulin-dependent form demonstrated a non-competitive inhibition between the two substrates, suggesting the presence of separate catalytic sites. This is in agreement with kinetic parameters and different thermal stabilities of cyclic AMP- and cyclic GMP-hydrolysing activities. Furthermore, the relevant change in s value, depending on the absence or presence of Ca2+ and calmodulin, suggested that the enzyme is composed of subunits, which aggregate in the presence of the activator. A model for catalytic site composition and reciprocal interaction is also proposed.     

Cyclic nucleotide phosphodiesterases of Hyalophora cecropia silkmoth fat body.

Two soluble forms of 3':5'-cyclic-nucleotide phosphodiesterase (o':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the larval fat body of the silkmoth Hyalophora cecropia. These differ in elution profile on Sephadex G-200, solubility in ammonium sulfate, metal ion requirements and kinetic properties. Phosphodiesterase I has Km values of 11 muM and 1.8 muM for cyclic AMP and cyclic GMP, respectively, has 5-fold greater maximal activity with cyclic AMP than with cyclic GMP, and is activated by Mg2+ and Co2+, and inhibited by EDTA. phosphodiesterase II has Km values of 625 muM and 125 muM for cyclic AMP and cyclic GMP, respectively, has similar maximal activity with both substrates, and is not activated by divalent metal ions or inhibited by EDTA. Cyclic nucleotides and methylxanthines competitively inhibit both enzymes. Phosphodiesterase is found in both soluble and particulate fractions of homogenates. Total activity is highest during the larval stage of the insect, drops markedly following pupation, and rises again during pharate adult development.     

Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate.

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.     

Reconsideration of the multiple cyclic nucleotide phosphodiesterases in bovine heart.

DEAE-cellulose chromatography demonstrated the presence of three peaks of cyclic nucleotide phosphodiesterase activity in the hearts of cattle during the summer and only two peaks during exposure to freezing temperatures. The hydrolysis of 10^?6M cyclic AMP by peak II, the variable activity, was stimulated 160% by 10^?6M cyclic GMP and was inhibited by chelation of Ca²⁺. Peak II activity was not a distinct enzyme but rather a mixture of activator-dependent phosphodiesterase, phosphodiesterase activator and type II cyclic AMP-dependent protein kinase.