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-competitively 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 muM Ca2+ and phosphodiesterase activator with Ki values of approximately 2 muM for both substrates. CuCl2 inhibition was also non-competitive with Mg2+, Ca2+ and phosphodiesterase activator. Dialysis demonstrated that CuCl2 inhibition is 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 of 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 muM. 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.     

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.     

Amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase from bovine heart

The complete amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase (cGS-PDE) of bovine heart has been determined by analysis of five digests of the protein; placement of the C-terminal 330 residues has been confirmed by interpretation of the corresponding partial cDNA clone. The holoenzyme is a homodimer of two identical N alpha-acetylated polypeptide chains of 921 residues, each with a calculated molecular weight of 103,244. The C-terminal region, residues 613-871, of the cGS-PDE comprises a catalytic domain that is conserved in all phosphodiesterase sequences except those of PDE 1 from Saccharomyces cerevisiae and a secreted PDE from Dictyostelium. A second conserved region, residues 209-567, is homologous to corresponding regions of the alpha and alpha' subunits of the photoreceptor phosphodiesterases. This conserved domain specifically binds cGMP and is involved in the allosteric regulation of the cGS-PDE. This regulatory domain contains two tandem, internal repeats, suggesting that it evolved from an ancestral gene duplication. Common cyclic nucleotide binding properties and a distant structural relationship provide evidence that the catalytic and regulatory domains within the cGS- and photoreceptor PDEs are also related by an ancient internal gene duplication.     

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.     

Analysis of the kinetics of cyclic AMP hydrolysis by the cyclic GMP-stimulated cyclic nucleotide phosphodiesterase

The kinetics of cAMP hydrolysis by the purified calf liver cGMP-stimulated cyclic nucleotide phosphodiesterase were analyzed in the absence or presence of a number of competitive inhibitors of the methylxanthine type according to a two-site competitive model for allosteric enzymes. Methylxanthines were also classified by graphical analysis of classical competition kinetics at saturating cAMP. This treatment yielded Km/KI ratios which estimated the relative effectiveness of the binding of substrate and inhibitors to the "high affinity" (ES complex) state without establishing individual equilibrium-binding constants of cAMP and inhibitors for specific enzyme states. Individual binding constants for substrate and inhibitors were estimated directly by fitting primary data to the rate equation for the two-site competitive model. The equilibrium dissociation constants for cAMP to the "high" (KS) and "low affinity" (AKS) states were 2.4 +/- 0.8 and 410 +/- 140 microM, respectively. Dissociation constants for various inhibitors to the high (BKI) and low affinity (KI) states were also estimated. The ratio KS/BKI, which directly compared the equilibrium-binding constants of substrate and inhibitors to the high affinity state (ES complex), was in excellent agreement with Km/KI ratios derived from graphical analysis. Whereas a number of the methylxanthine analogues were more effective or as effective as cAMP in binding to the low affinity or "ligand-free" state, only isobutylmethylxanthine was effective as cAMP in binding to the high affinity state (1-methyl-3-isopropylxanthine, and 1,3-dipropylxanthine were somewhat less effective). These findings suggested that allosteric transitions might alter the topography of specific hydrophobic domains at cyclic nucleotide-binding sites and that structural determinants were more stringent for binding to the high affinity state than to the low affinity state.     

A continuous fluorescence assay for cyclic nucleotide phosphodiesterase hydrolysis of cyclic GMP

The fluorescent 2'-methylanthraniloyl derivative of cyclic GMP undergoes a 45% decrease in fluorescence when it is cleaved by brain phosphodiesterase in the presence of calmodulin. This fluorescence decrease is dependent upon calcium, calmodulin, and phosphodiesterase, and correlates well (r = 0.996) with the disappearance of substrate as monitored by high-performance liquid chromatography. The Kd values determined by this fluorescence method and HPLC suggest that cyclic GMP and its fluorescent derivative exhibit similar kinetic parameters in their hydrolysis.     

Effects of phosphodiesterase inhibitors, imidazole and phosphate on cyclic CMP phosphodiesterase are different from those on cyclic AMP and cyclic GMP phosphodiesterases.

The effects of various agents on the newly identified cyclic CMP phosphodiesterase (C-PDE) in crude extracts of a number of rat tissues and on the enzyme partially purified from the rat liver were examined. Papaverine and 1-methyl-3-isobutylxanthine were without effects on C-PDE at concentrations that inhibited up to 90% of cyclic AMP phosphodiesterase (A-PDE) and cyclic GMP phosphodiesterase (G-PDE) activities. When assayed using 1 micron substrates, theophylline inhibited C-PDE to a lesser extent than A-PDE and G-PDE. 2'-Deoxy cyclic AMP (specific A-PDE inhibitor) and 2'-deoxy cyclic GMP (specific G-PDE inhibitor) were relatively poor and non-specific inhibitors for C-PDE. Imidazole, while augmenting the high Km A-PDE and G-PDE from the liver but not from the heart, was without effect on the liver C-PDE but stimulated the heart C-PDE. Potassium phosphate was more specific in inhibiting C-PDE than A-PDE and G-PDE. The present findings suggest that C-PDE represents a potential site of specific pharmacological regulations, and that C-PDE may be a separate enzyme distinguishable from the purine cyclic nucleotide class of phosphodiesterases.     

Cyclic GMP stimulation and inhibition of cyclic AMP phosphodiesterase from thymic lymphocytes

A particulate preparation of cyclic AMP phosphodiesterase from rat thymic lymphocytes exhibited two apparent Km's at 0.9×10⁻⁶M and 8.0×10⁻⁶M. The enzyme with the higher Km was stimulated by cyclic GMP by a mechanism involving an increase in the Vmax of the enzyme with no change in the Km. Cyclic GMP competitively inhibited the enzyme with the low apparent Km which had a Ki for cyclic GMP of 4×10⁻⁵M. The modulation of cyclic AMP phosphodiesterase activity by cyclic GMP in the control of cyclic AMP-mediated lymphocyte proliferation is discussed.     

Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities of rat mammary tissue.

Cyclic nucleotide phosphodiesterase activity in mammary tissue from rats in midlactation was resolved by DEAE-cellulose chromatography into three functionally distinct fractions: a Ca2+/calmodulin-stimulated cyclic GMP phosphodiesterase, a cyclic GMP-stimulated low-affinity cyclic nucleotide phosphodiesterase, and a high-affinity cyclic AMP-specific phosphodiesterase. The absolute activities and relative proportions of high- and low-affinity enzymes resemble those found, for example, in liver, as distinct from those in excitable tissues. Three functional characteristics are described which are peculiar to mammary-tissue phosphodiesterases. Firstly, the concentration of free Ca2+ required to achieve half-maximal activation of the Ca2+/calmodulin-stimulated phosphodiesterase is somewhat higher than for the analogous enzyme in other tissues; secondly, the activity of this enzyme towards cyclic AMP relative to that towards cyclic GMP is unusually low, and thirdly, the low-affinity cyclic nucleotide phosphodiesterase is inhibited by low concentrations of free Ca2+.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (K_m 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (K_m 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg²⁺. Mn²⁺ (3mm) was as effective as Mg²⁺ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca²⁺ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg²⁺ was 3mm. At concentrations of Mg²⁺ between 0.1 and 1mm, 1mm-Ca²⁺ was activatory, and at concentrations of Mg²⁺ below 0.1mm, 1mm-Ca²⁺ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.     

Specificity of cyclic GMP activation of a multi-substrate cyclic nucleotide phosphodiesterase from rat liver

Phosphoprotein phosphatase IA, which represents the major glycogen synthase phosphatase activity in rat liver cytosol, has been purified to apparent homogeneity by chromatography on DEAE-cellulose, histone - Sepharose-4B and Sephadex G-100. The molecular weight of the purified enzyme was 40 000 by gel filtration and 48 000 by sodium dodecyl sulfate gel electrophoresis, Phosphatase IA is therefore a monomeric protein. When treated with 80% ethanol at room temperature, phosphatase IA underwent an inactivation which was totally prevented by 2 mM MgCl2. Catalytically, phosphatase IA has a preference for glycogen synthase D compared with phosphatases IB and II and obligatorily requires Mg2+ or Mn2+ for activity. Maximum activity was attained at 5 mM MgCl2. Since Mg2+ does not activate other phosphoprotein phosphatases in rat liver cytosol, we propose the term 'Mg2+-dependent glycogen synthase phosphatase' for phosphatase IA.     

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.     

Isolation of cyclic AMP and cyclic GMP by thin-layer chromatography. Application to assay of adenylate cyclase, guanylate cyclase, and cyclic nucleotide phosphodiesterase

A procedure is described for isolation of cAMP and cGMP by thin-layer chromatography on polyethylenimine cellulose. Chromatographs are developed (descending) twice in the same direction with two different solvents. This procedure separates cAMP and cGMP from other radioactive metatolites of [³H] or [¹⁴C] ATP or GTP. Application of this isolation method to assay of adenylate cyclase, (EC 4.6.1.1), guanylate cyclase (EC 4.6.1.2), and cyclic nucleotide phosphodiesterase (EC 3.1.4.17) has proven convenient and provides results of unusual quality.     

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.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a pea a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2–3 fold increase at 15–30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E₂ also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise incyclic AMP. These results suggest that the relative amount of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

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).