Calcium-dependent cyclic nucleotide phosphodiesterase. Inhibition of basal activity by heat-stable factors from rat cerebrum.

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca2+-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca2+-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in Km for cyclic GMP and a decrease in V. In the presence of calcium ions and purified activator protein, the Ca2+-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca2+-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was minimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca2+-independent activation of Ca2+-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca2+-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca2+-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phoshodiesterase activity.     

Effect of lipids on the binding of calmodulin with cyclic nucleotide phosphodiesterase

The effects of various lipids on calmodulin interaction with Ca-dependent phosphodiesterase were investigated. Palmitic, myristic and stearic acids increased the enzyme activity; the degree of the enzyme activation by calmodulin was decreased thereby. Oleic acid produced a weak activating effect on phosphodiesterase but completely blocked calmodulin action. The effects of the fatty acids under study were reversible, the activation constant was equal to 10(-4)-5 X 10(-4) M. In the presence of Ca2+ phosphoinositides and fatty acids changed the fluorescence intensity of dansyl-labelled calmodulin; in the absence of Ca2+ the lipids did not affect protein fluorescence. The lipids had no influence on the protein affinity for Ca2+. During chromatography of phosphodiesterase on calmodulin-Sepharose the enzyme was eluted from the column both in the presence of EGTA and palmitic acid. It was concluded that fatty acids prevent the formation of the calmodulin - phosphodiesterase complex. This effects may both be due to the lipid binding to the enzyme and to calmodulin.     

Purification of a Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart by specific interaction with its Ca2+-dependent modulator protein.

A Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart can be eluted from a DEAE-cellulose column either in the free form by buffers containing 0.1 mM ethylene glycol bis(beta-aminoethyl ether)N-N,N'N'-tetraacetic acid (EGTA) or as a complex of the enzyme with its protein modulator by buffers containing 0.01 mM CaCl2. A purification procedure based primarily on the significantly different affinity of the two forms of the enzyme for DEAE-cellulose was developed for the purification of the enzyme from bovine heart. The procedure involves ammonium sulfate fractionation, three chromatographic steps on DEAE-cellulose, and gel filtration on Sephadex G-200 with a 5000-fold purification over the crude extract. The purified enzyme has a specific activity of 120 mumol of cAMP/mg/min, can be activated 5-fold by Ca2+, but is only 80% pure as judged by analytical disc gel electrophoresis. The purified enzyme is unstable but can be stabilized by addition of Ca2+ and the protein modulator; this is in contrast to the less pure preparations of Ca2+-activatable phosphodiesterase which are destabilized by the protein modulator in the presence of Ca2+.     

Purification of cyclic 3',5'-nucleotide phosphodiesterase inhibitory protein by affinity chromatography on activator protein coupled to Sepharose.

The Ca2+-dependent, reversible, interaction of cyclic adenosine 3',5'-monophosphate (cAMP) phosphodiesterase with its activator has been used to purify the enzyme by affinity chromatography. Activator-dependent cAMP phosphodiesterase is only a minor component of the proteins specifically adsorbed in the presence of Ca2+ by the Ca2+-dependent activator protein coupled to Sepharose and subsequently released by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The major protein component can be partially resolved from the enzyme by gel filtration on Sephadex G-200. This protein has been purified to apparent homogeneity and shown to be composed of two polypeptide chains with molecular weights of 61,000 and 15,000 respectively. This protein is, by itself, devoid of phosphodiesterase activity and inhibits the activation of cAMP phosphodiesterase by its activator without affecting the basal activity. Thus, activation of cAMP phosphodiesteriase by the Ca2+-dependent activator protein may be controlled by interactions with yet a third component of the enzyme complex.     

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+.     

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.     

Calcium-dependent protein modulator of cyclic nucleotide phosphodiesterases from mouse epidermis.

1. A heat-stable modulator protein was partially purified from mouse epidermis. The protein stimulated modulator-depleted cyclic AMP phosphodiesterase from bovine brain in the presence of Ca2+. 2. DEAE-cellulose chromatography of epidermal extracts demonstrated the presence of two main phosphodiesterase activities that hydrolysed both cyclic AMP and cyclic GMP. A minor peak was eluted between 0.1 and 0.3 M-sodium acetate and a major peak was eluted between 0.3 and 0.45 M-sodium acetate. 3. Cyclic AMP phosphodiesterase activity eluted at low salt concentrations was markedly activated by the epidermal modulator protein in the presence of Ca2+. Storage of the enzyme led to a decrease in its sensitivity to the protein modulator. 4. Treatment of mouse skin with the tumour promoter 12-O-tetradecanoylphorbol 13-acetate, which leads to an increase in epidermal cyclic nucleotide phosphodiesterase activity, did not alter the amount of modulator present in soluble epidermal extracts. The tumour promoter decreased the amount of modulator extractable from particulate epidermal preparations with Triton X-100.     

Multiple forms of cyclic nucleotide phosphodiesterase in a murine adrenal cortex cell line (Y-1)

Murine adrenal cortex tumor Y-1 cells contained both soluble and particulate forms of cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotide hydrolase, EC 3.1.4.17). The soluble forms of the enzyme comprised 80% of total cellular phosphodiesterase activity. The soluble enzyme(s) hydrolyzed both cyclic AMP and cyclic GMP, with apparent Km values of 125 and 30 microM, respectively. Soluble cyclic AMP phosphodiesterase showed marked inhibition by the calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and the anticalmodulin drugs, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium. No alteration in soluble cyclic GMP phosphodiesterase activity was observed when cyclic AMP was added to the assay. Resolution of the soluble enzymatic activity by DEAE-cellulose chromatography in the presence of calcium showed two peaks of phosphodiesterase activity. Further purification of one of these peaks on DEAE-cellulose in the presence of EGTA yielded a phosphodiesterase activity peak that was stimulated fivefold by calmodulin. The particulate form of the enzyme hydrolyzed both cyclic AMP anc cyclic GMP; the apparent Km values for these substrates were similar (90 and 100 microM, respectively). Hydrolysis of cyclic GMP by the particulate enzyme was inhibited by cyclic AMP in a concentration-dependent manner with an apparent half-maximal inhibitory concentration of 100 microM. The particulate form of phosphodiesterase was not inhibited by EGTA or anticalmodulin drugs.     

Calcium-dependent cyclic nucleotide phosphodiesterase inhibition of basal activity by heat-stable factors from rat cerebrum

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca²⁺-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca²⁺-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in K_m for cyclic GMP and a decrease in $\text{V}$. In the presence of calcium ions and purified activator protein, the Ca²⁺-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca²⁺-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was mimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca²⁺-independent activation of Ca²⁺-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca²⁺-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca²⁺-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phosphodiesterase activity.     

A novel phosphodiesterase I from the soluble fraction of erythrocytes

A previously unrecognized erythrocyte phosphodiesterase I with activity against thymidine-5'-monophospho-p-nitrophenyl ester is described. The enzyme is present in the soluble fraction of the erythrocyte, and was purified about 500-fold by chromatography using DEAE-cellulose, followed by gel chromatography with Sephadex G-200. Erythrocyte phosphodiesterase I has a molecular weight of about 70 000, when fully active as a monomer. Its pI is 5.4 and the pH optimum is 8.5. The Km value for thymidine-5'-monophospho-p-nitrophenyl ester is rather high, about 4 mmol/l. The enzyme has a barely detectable nucleotide pyrophosphatase activity. It is extremely sensitive to SH-inhibitors such as N-ethyl-maleimide, p-chloromercuribenzoate and disulphides (a reversible 50% inhibition was obtained by cystamine, 0.01 mmol/l). It is a metalloenzyme with loosely bound metal, and is stimulated by Mg2+. This activation by Mg2+ is counteracted by Zn2+. Gel chromatography revealed that the enzyme is a monomer in the presence of Mg2+. When inhibited by Zn2+, it forms polymers that can be reconverted to the monomer by thiols. All of the above properties of the erythrocyte enzyme support the conclusion that it is different from plasma membrane phosphodiesterase I (oligonucleate 5'-nucleotidohydrolase, EC 3.1.4.1).     

Modulation of lyso-platelet-activating factor: acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions

The enzyme lyso-platelet-activating factor: acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca2+ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca2+. The effect of Ca2+ was on the apparent Km of the enzyme for the substrate acetyl-CoA without showing any significant effect on the Vmax of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca2+ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca2+, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca2+ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.     

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+.     

Cyclic 3':5'-nucleotide phosphodiesterase. Stimulation of bovine brain cytoplasmic enzyme by lysophosphatidylcholine.

Brain cytoplasmic cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) requires an endogenous Ca2+-binding protein for ful activity. We now show that lysophosphatidylcholine also effectively enhances activator-deficient phosphodiesterase activity. Stimulation by both ligands was immediate and reversible; both rendered the enzyme more thermally labile, decreased the energy of activation, and increased the Vmax of phosphodiesterase without affecting its apparent Km for adenosine 3'5'-monophosphate. However, the cofactor requirements of the two ligands were different. Although the protein activator gave a greater stimulation than lysophosphatidylcholine, the simultaneous presence of the two gave a stimulation comparable to lysophosphatidylcholine, suggesting that the effect of the latter was predominant. Phosphodiesterase was also stimulated by oleic acid, cardiolipin, and phosphatidylinositol, albeit to a less extent.     

Preparation of an enzymatically active cross-linked complex between brain cyclic nucleotide phosphodiesterase and 3-(2-pyridyldithio)propionyl-substituted calmodulin

Cyclic nucleotide phosphodiesterase (0.07 nM) was activated by near stoichiometric concentrations of [3-(2-pyridyldithio)propionyl]calmodulin (PDP-CaM) after initial incubation of these proteins at 200-fold higher concentrations; activity in assays with EGTA was 80% of that in the presence of Ca2+. The enzyme incubated with native calmodulin under identical conditions required approximately 1 nM for half-maximal activation, and no activation was observed in the absence of calcium. These data suggested formation of a covalent complex between phosphodiesterase and PDP-CaM. On high-performance gel-permeation chromatography in the presence of metal chelators, the complex appeared considerably larger than the native enzyme. Incubation of phosphodiesterase with the thiolated (inactivated) form of PDP-CaM did not change its chromatographic behavior, indicating that reactive sulfhydryl groups were involved in complex formation. Although the total activities recovered from chromatography were not significantly different, maximal activation of PDP-CaM-phosphodiesterase complex was only approximately 20%, whereas the control enzyme was activated 6-8-fold by Ca2+ plus calmodulin. Kinetics of cGMP hydrolysis in the presence of EGTA by the isolated complex differed from those of control enzyme but were indistinguishable from those of control enzyme assayed with saturating Ca2+ and CaM. The calmodulin antagonists W-7 and trifluoperazine had relatively little effect on activity of the PDP-CaM-phosphodiesterase complex. Incubation of the complex with dithiothreitol dramatically increased its Ca2+ and calmodulin responsiveness, suggesting that reduction of the disulfide cross-link released phosphodiesterase from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Partial purification and properties of guanosine 3':5'-monophosphate-dependent protein kinase from pig lung.

Guanosine 3':5'-monophosphate(cyclic GMP)-dependent protein kinase which catalyzes the phosphorylation of histone was purified about 200-fold from the soluble fraction of pig lung by pH 5.5 precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. The apparent Ka values for guanosine 3':5'-monophosphate and adenosine 3':5'-monophosphate were determined to be about 17 and 360 nM, respectively. Mg2+ was essential for the activity exhibiting biphasic stimulation behavior and neither Mn2+ nor Ca2+ could substitute for Mg2+. However, these divalent ions markedly inhibited the protein kinase activity stimulated by cyclic GMP in the presence of Mg2+.     

Phosphorylation and characterization of bovine heart calmodulin-dependent phosphodiesterase.

Calmodulin-dependent phosphodiesterase was purified to apparent homogeneity from the total calmodulin-binding fraction of bovine heart in a single step by immunoaffinity chromatography. The isolated enzyme had significantly higher affinity for calmodulin than the bovine brain 60-kDa phosphodiesterase isozyme. The cAMP-dependent protein kinase was found to catalyze the phosphorylation of the purified cardiac calmodulin-dependent phosphodiesterase with the incorporation of 1 mol of phosphate/mol of subunit. The phosphodiesterase phosphorylation rate was increased severalfold by histidine without affecting phosphate incorporation into the enzyme. Phosphorylation of phosphodiesterase lowered its affinity for calmodulin and Ca2+. At constant saturating concentrations of calmodulin (650 nM), the phosphorylated calmodulin-dependent phosphodiesterase required a higher concentration of Ca2+ (20 microM) than the nonphosphorylated phosphodiesterase (0.8 microM) for 50% activity. Phosphorylation could be reversed by the calmodulin-dependent phosphatase (calcineurin), and dephosphorylation was accompanied by an increase in the affinity of phosphodiesterase for calmodulin.     

Photolytic studies on the carbon monoxide complex of sulphaemoglobin.

Salivary-gland homogenates contain 5-hydroxytryptamine-stimulated adenylate cyclase. Half-maximal stimulation was obtained with 0.1 microM-5-hydroxytryptamine in the presence of added guanine nucleotides. Gramine antagonized the stimulation of cyclase caused by 5-hydroxytryptamine. In the presence of hormone, guanosine 5'-[gamma-thio]triphosphate produced a marked activation of adenylate cyclase activity. Stimulation of adenylate cyclase by forskolin or fluoride did not require the addition of guanine nucleotides or hormone. In the presence of EGTA, Ca2+ produced a biphasic activation of cyclase activity. Ca2+ at 1-100 microM increased activity, whereas 2000 microM-Ca2+ inhibited cyclase activity. The neuroleptic drugs trifluoperazine and chlorpromazine non-specifically inhibited adenylate cyclase activity even in the absence of Ca2+. The cyclic AMP phosphodiesterase activity in homogenates was not affected by Ca2+ or exogenous calmodulin. This enzyme was also inhibited by trifluoperazine in the absence of Ca2+. These results indicate that Ca2+ elevates adenylate cyclase activity, but had no effect on cyclic AMP phosphodiesterase of salivary-gland homogenates.     

Regulation of cyclic GMP phosphodiesterase in the submandibular gland of adult rat

Cyclic GMP phosphodiesterase was investigated in the submandibular gland of the adult rat to elucidate the regulatory mechanisms of cGMP concentration in this gland. Ca2+ sensitivity was easily demonstrated as in other tissues using EGTA in the buffer for elution from DEAE-cellulose. The presence of inhibitor proteins for basal and calmodulin-activated portions of Ca2+-dependent phosphodiesterase was suggested. The inhibitor for the basal activity of Ca2+-dependent phosphodiesterase was deemed to be a heat-labile protein, which decreased Vmax, but had no effect on the Km value for cGMP. The presence of more than one kind of inhibitor for the calmodulin-activated portion of the Ca2+-dependent phosphodiesterase was also suggested. One of these, which was not absorbed on DEAE-cellulose, was a heat-labile protein which caused an increase of Km for cGMP, but no change of Vmax.     

The dependence on Ca2+ of the guanine-nucleotide-activated polyphosphoinositide phosphodiesterase in neutrophil plasma membranes.

The requirement for Ca2+ for the activation of polyphosphoinositide phosphodiesterase was studied with the guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). Levels of Ca2+ that pertain in unstimulated neutrophils (100 nM) are obligatory for the full expression of enzyme activity stimulated with GTP gamma S. Reduction of Ca2+ to 1 nM leads to inhibition. Increasing the level of Ca2+ from 100 nM to 1000 nM does not alter enzyme activity. Guanosine 5'-[beta-thio]diphosphate (GDP beta S) does not stimulate the phosphodiesterase but is an effective inhibitor of activation by GTP gamma S. Ca2+ in the millimolar range can also activate the phosphodiesterase alone and this is not inhibited by GDP beta S. It is also shown that Sr2+ in the millimolar range can stimulate enzyme activity similarly to Ca2+.