Concanavalin A stimulates the Rolipram-sensitive isoforms of cyclic nucleotide phosphodiesterase in rat thymic lymphocytes

Pretreatment of rat thymic lymphocytes with Concanavalin A induced a very early (30 min) and substantial increase (+90%) of the soluble cAMP phosphodiesterase activity. The crude cytosolic phosphodiesterase activity of rat thymocytes could reproducively be resolved by Mono-Q ion exchange high performance liquid chromatography into four separate phosphodiesterase peaks: a cGMP-stimulated, two cAMP-specific Rolipram-sensitive and a cGMP-inhibited cardiotrope-sensitive peaks. Concanavalin A stimulated very specifically the activity of the two predominant cAMP-specific Rolipram sensitive peaks whereas it only slightly modified the cGMP-stimulated and the cGMP-inhibited forms. The present results strongly suggest that the Rolipram-sensitive cAMP PDE activity may play a key role in the control and regulation of mitogen-induced thymocyte proliferation.     

Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives

Cyclic nucleotide derivatives have been used as a tool to characterize distinct catalytic sites on phosphodiesterase enzyme forms: the cGMP-stimulated enzyme from rat liver and the calmodulin-sensitive enzyme from rat or bovine brain. Under appropriate assay conditions, the analogues showed linear competitive inhibition with respect to cAMP (adenosine 3',5'-monophosphate) as substrate. The inhibition sequence of the fully activated cGMP-stimulated phosphodiesterase was identical to the inhibition sequence of the desensitized enzyme, i.e. the enzyme which has lost its ability to be stimulated by cGMP. The inhibition pattern could, therefore, not be attributed to competition with cGMP at an allosteric-activating site. Also, the inhibition sequence of the calmodulin-sensitive phosphodiesterase was maintained whether activity was basal or fully stimulated by calmodulin. When cAMP and cGMP, with identical chemical ligands substituted at the same position, were compared as inhibitors of the calmodulin-sensitive phosphodiesterase, the cGMP analogues were always the more potent suggesting that, for that enzyme, the catalytic site was sensitive to a guanine-type cyclic nucleotide structure. Comparing the two phosphodiesterases, it was possible to establish both similar and specific inhibitor potencies of cyclic nucleotide derivatives. In particular, the two enzymes exhibited large differences in analogue specificity modified at C-6, 6-chloropurine 3',5'-monophosphate or purine 3',5'-monophosphate.     

Identification and characterization of two unusual cGMP-stimulated phoshodiesterases in dictyostelium

Recently, we recognized two genes, gbpA and gbpB, encoding putative cGMP-binding proteins with a Zn(2+)-hydrolase domain and two cyclic nucleotide binding domains. The Zn(2+)-hydrolase domains belong to the superfamily of beta-lactamases, also harboring a small family of class II phosphodiesterases from bacteria and lower eukaryotes. Gene inactivation and overexpression studies demonstrate that gbpA encodes the cGMP-stimulated cGMP-phosphodiesterase that was characterized biochemically previously and was shown to be involved in chemotaxis. cAMP neither activates nor is a substrate of GbpA. The gbpB gene is expressed mainly in the multicellular stage and seems to encode a dual specificity phosphodiesterase with preference for cAMP. The enzyme hydrolyses cAMP approximately 9-fold faster than cGMP and is activated by cAMP and cGMP with a K(A) value of approximately 0.7 and 2.3 microM, respectively. Cells with a deletion of the gbpB gene have increased basal and receptor stimulated cAMP levels and are sporogeneous. We propose that GbpA and GbpB hydrolyze the substrate in the Zn(2+)-hydrolase domain, whereas the cyclic nucleotide binding domains mediate activation. The human cGMP-stimulated cAMP/cGMP phosphodiesterase has similar biochemical properties, but a completely different topology: hydrolysis takes place by a class I catalytic domain and GAF domains mediate cGMP activation.     

Kinetic properties and regulation of cyclic nucleotide phosphodiesterases in lymphoid cells

cGMP-dependent cyclic nucleotide phosphodiesterases have been isolated from spleen lymphocytes and the whole mice spleen and shown to possess identical properties. Two structure analogues of cAMP and cGMP, viz. N6,O2'-dibutyryl-cAMP and N2,O2'-dibutyryl-cGMP, were used to investigate the properties of the phosphodiesterase and found to inhibit hydrolysis of both cAMP and cGMP. This inhibition did not affect the cGMP activation constant. Existence of two different centres of catalytic and regulatory types in cGMP-stimulated phosphodiesterase is suggested.     

The role of protein phosphorylation in the regulation of cyclic nucleotide phosphodiesterases

The cyclic nucleotide phosphodiesterases constitute a complex superfamily of enzymes responsible for catalyzing the hydrolysis of cyclic nucleotides. Regulation of cyclic nucleotide phosphodiesterases is one of the two major mechanisms by which intracellular cyclic nucleotide levels are controlled. In many cases the fluctuations in cyclic nucleotide cAMP-specific, calmodulin-stimulated and cGMP-binding phosphodiesterases have been demonstrated to be substrates for protein kinases. Here we review the evidence that hormonally responsive phosphorylation acts to regulate cyclic nucleotide phosphodiesterases. In particular, the cGMP-inhibited phosphodiesterases, which can be phosphorylated by at least two different protein kinases, are activated as a result of phosphorylation. In contrast, phosphorylation of the calmodulin-stimulated phosphodiesterases, which coincides with, a decreased sensitivity to activation by calmodulin, results in decreased phosphodiesterase activity.     

Dunce mutants of Drosophila melanogaster: mutants defective in the cyclic AMP phosphodiesterase enzyme system

The cyclic AMP and cyclic GMP phosphodiesterase activities present in flies of six mutant strains of the dunce gene and in the parent wild-type strains are characterized. All of the mutants exhibit aberrant cyclic AMP metabolism. The mutant strains dunceM14, dunceM11, and dunceML appear to be amorphic, because they completely lack the cAMP-specific phosphodiesterase normally present in adult flies. These strains exhibit extremely high levels of cAMP. The mutant strains dunce1, dunce2, and dunceCK are hypomorphic and exhibit reduced levels of the cAMP-specific phosphodiesterase. These strains exhibit less marked increases in cAMP content compared with the three amorphic strains. The dunce2 strain possesses a residual enzyme activity that exhibits anomalous kinetics compared with those of the normal enzyme. The possibility that the dunce locus is the structural gene for the cAMP-specific phosphodiesterase is discussed.     

Adrenal medullary cyclic nucleotide phosphodiesterase: lack of activation by the calcium-dependent regulator.

Calcium-dependent regulator, a calcium-binding protein isolated from brain and adrenal medulla, has been shown to activate a brain calcium-sensitive cyclic nucleotide phosphodiesterase. To determine if this protein has the same role in the adrenal medulla, the cyclic nucleotide phosphodiesterase of adrenal medulla was characterized. Neither crude nor partially purified adrenal medullary phosphodiesterase was inhibited by EGTA or stimulated by calcium and the calcium-dependent regulator, whereas similar brain preparations displayed sensitivity to these agents. As the calcium-dependent regulator does not appear to stimulate adrenal medullary cyclic nucleotide phosphodiesterase activity, alternate roles of this protein in adrenal medulla are suggested.     

The identification and characterization of two cyclic nucleotide phosphodiesterases from bovine adrenal medulla

Two soluble cyclic nucleotide phosphodiesterase activities, designated Peak I (Mr = 216,000) and Peak II (Mr = 230,000), have been isolated from bovine adrenal medulla by DEAE-cellulose chromatography. Peak I has Ca2+-independent, cGMP-specific phosphodiesterase activity and Peak II has cGMP-stimulated cyclic nucleotide phosphodiesterase activity. Peak I hydrolyzes cGMP with hyperbolic kinetics and demonstrates a Km of 23 microM. Peak II hydrolyzes cGMP with hyperbolic kinetics but hydrolyzes cAMP with slightly sigmoidal kinetics and demonstrates Km values of 54 +/- 0.7 microM cGMP and 38 +/- 6 microM cAMP. Cyclic AMP and cGMP are competitive inhibitors of each other's hydrolysis, suggesting that these nucleotides may be hydrolyzed at the same catalytic site. Micromolar concentrations of cGMP cause a 5-fold stimulation of the hydrolysis of subsaturating concentrations of cAMP by the Peak II phosphodiesterase. Half-maximal activation occurs at 0.5 microM cGMP and the result of activation is a decrease in the apparent Km for cAMP. Stimulation of the hydrolysis of subsaturating concentrations of cGMP by cAMP was also detected; however, cAMP is a less potent activator of the enzyme than cGMP. Cyclic AMP causes a 1.5-fold stimulation of cGMP hydrolysis and half-maximal activation occurs at 2.5 microM cAMP.     

Characterization of the soluble cyclic nucleotide phosphodiesterases in Xenopus laevis oocytes. Evidence for a calmodulin-dependent enzyme

Cyclic nucleotide phosphodiesterase activities (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the 40,000 X g supernatant fraction of homogenates of Xenopus laevis oocytes. In the supernatant, the ratio of the specific activity of cyclic AMP phosphodiesterase to that of cyclic GMP phosphodiesterase was 1.1 at the 1 micro substrate level. Two phosphodiesterase forms were isolated by centrifugation on sucrose gradient: a 3-4 S form hydrolyzing specificity cyclic AMP and a 6-7 S form hydrolyzing both cyclic nucleotides (cyclic AMP and cyclic GMP). The activity of the 6-7 S phosphodiesterase was characterized by its activation by 0.1 micro M calmodulin purified from beef pancreas in the presence of 50 micro M CA2+. The calmodulin dependence of this form was completely abolished in the presence of 1 mM ethyleneglycobis(beta-aminoethyl ether)-N-N,N',N'-tetraacetic acid (EGTA). Trifluoperazine at 0.1 mM inhibited both the freshly prepared crude enzyme and the partially purified 6-7 S form. On the other hand, no effect of cyclic GMP at 3 micro M was observed on cyclic AMP hydrolysis in the case of the supernatant or that of the partially purified phosphodiesterases. These data show the presence of a calmodulin-dependent phosphodiesterase in the soluble fraction of X. laevis oocytes.     

Purification, characterization and production of rabbit antibodies to rat liver particulate, high-affinity, cyclic AMP phosphodiesterase

The cyclic nucleotide phosphodiesterase (EC 3.4.16) activities of a rat liver particulate fraction were analyzed after solubilization by detergent or by freeze-thawing. Analysis of the two extracts by DEAE-cellulose chromatography revealed that they contain different complements of phosphodiesterase activities. The detergent-solubilized extract contained a cyclic GMP phosphodiesterase, a low affinity cyclic nucleotide phosphodiesterase whose hydrolysis of cyclic AMP was activated by cyclic GMP and a high affinity cyclic AMP phosphodiesterase. The freeze-thaw extract contained a cyclic GMP phosphodiesterase and two high affinity cyclic AMP phosphodiesterase, but no low affinity cyclic nucleotide phosphodiesterase. The cyclic AMP phosphodiesterase activities from the freeze-thaw extract and from the detergent extract all had negatively cooperative kinetics. One of the cyclic AMP phosphodiesterases from the freeze-thaw extract (form A) was insensitive to inhibition by cyclic GMP; the other freeze-thaw solubilized cyclic AMP phosphodiesterase (form B) and the detergent-solubilized cyclic AMP phosphodiesterase were strongly inhibited by cyclic GMP. The B enzyme appeared to be converted into the A enzyme when the particulate fraction was stored for prolonged periods at -20 degrees C. The B form was purified extensively, using DEAE-cellulose, a guanine-Sepharose column and gel filtration. The enzyme retained its negatively cooperative kinetics and high affinity for both cyclic AMP and cyclic GMP throughout the purification, although catalytic activity was always much greater for cyclic AMP. Rabbit antiserum was raised against the purified B enzyme and tested via a precipitin reaction against other forms of phosphodiesterase. The antiserum cross-reacted with the A enzyme and the detergent-solubilized cyclic AMP phosphodiesterase from rat liver. It did not react with the calmodulin-activated cyclic GMP phosphodiesterase of rat brain, the soluble low affinity cyclic nucleotide phosphodiesterase of rat liver or a commercial phosphodiesterase preparation from bovine heart. These results suggest a possible interrelationship between the high affinity cyclic nucleotide phosphodiesterase of rat liver.     

Cyclic AMP-specific phosphodiesterase 4 inhibitors promote ABCA1 expression and cholesterol efflux.

ATP cassette binding protein 1 (ABCA1) controls the apolipoprotein-mediated cholesterol efflux pathway and determines plasma HDL levels. Although cAMP is known to promote ABCA1 expression and cholesterol efflux from cells, it has not been determined whether cyclic nucleotide phosphodiesterase (PDE) isoforms regulate this pathway. We show that rolipram and cilomilast, inhibitors of cAMP-specific PDE4, increase apolipoprotein A-I (apoA-I)-mediated cholesterol efflux up to 80 and 140% in human THP-1 and mouse J774.A1 macrophages, respectively, concomitant with an elevation of cAMP levels. The EC(50) value was estimated to be 1 to 2 microM for both inhibitors. Rolipram and cilomilast also increase ABCA1 protein expression in THP-1 and J774.A1 macrophages. Thus, PDE4 inhibitors cause parallel increases in cAMP levels, ABCA1 expression and apoA-I-mediated cholesterol efflux. PDE4 inhibitors may provide a novel strategy for the treatment of cardiovascular disease by mobilizing cholesterol from atherosclerotic lesions.     

Absence of an effect of the lithium-induced increase in cyclic GMP on the cyclic GMP-stimulated phosphodiesterase (PDE II). Evidence for cyclic AMP-specific hydrolysis

Chronic treatment of rats with LiCl is known to induce a decrease in cAMP, while this decrease has also been found to occur together with both a simultaneous increase in total cortical phosphodiesterase (PDE; EC 3.1.4.17) activity and a concomitant increase in cGMP. These studies have implicated an involvement of PDE in lithium (Li⁺) action and it has been suggested that cGMP and the cGMP-stimulated PDE may be instrumental in the observed effects of Li⁺ on cAMP. In this study, three isozymes of PDE were isolated and identified from rat cortex and their activity determined, together with simultaneous measurement of cAMP and cGMP, after chronic treatment with oral LiCl (0.35% m/m). Li⁺ treatment exerted profound effects on cyclic nucleotides in the cortex, inducing significant suppression of cAMP while increasing cGMP levels. However, the ion only induced a slight but insignificant increase in the activities of the three PDE isozymes. To confirm these observations, methylparaben (MPB), a drug demonstrating both an ability to induce a selective stimulation of cAMP-specific PDE and also to lower intracellular levels of cGMP, was co-administration orally (0.4% m/m) with Li⁺ over the same period. This combination emphasized certain actions of Li⁺ not noted with Li⁺ alone. MPB inhibited the Li⁺-induced increase in cGMP, yet did not prevent the ion from decreasing cAMP. However, the combination of Li⁺ and MPB engendered a synergistic 100% increase in the activity of the membrane-bound, cAMP-specific PDE, PDE IV. This combination also produced a significant suppression of cAMP, while no reduction in cGMP was observed. The data is indicative that Li⁺-induced suppression of cAMP does not appear to be related to an effect on the cGMP-dependent PDE II, and that the increases in cGMP and PDE induced by Li⁺ observed previously and in the present study are two unrelated events. Instead, the synergistic response of Li⁺ plus MPB on PDE IV, and the associated reduction of cAMP, indicate that Li⁺ may promote selective cAMP hydrolysis via an effect on membrane-bound forms of PDE. This effect of Li⁺ on PDE IV, as well as the reciprocal effects on cyclic nucleotide balance, may have important implications in explaining the antipsychotic actions of the ion.     

Cyclic nucleotide phosphodiesterase from carrot

A cyclic nucleotide phosphodiesterase has been isolated and partially purified from carrot tap-root tissue. The properties of this enzyme are very different from cyclic AMP phosphodiesterases found in mammalian cells. A dialyzable inhibitor of carrot cyclic nucleotide phosphodiesterase was also isolated from carrot tissue. Because the inhibitor behaved like inorganic phosphate on ion-exchange chromatography and inhibited the enzyme in proportion to its inorganic phosphate content, the inhibitor was tentatively identified as inorganic phosphate.     

PDE7A1, a cAMP-specific phosphodiesterase, inhibits cAMP-dependent protein kinase by a direct interaction with C

The N-terminal regulatory region of the high affinity cAMP-specific phosphodiesterase, PDE7A1, contains two copies of the cAMP-dependent kinase (PKA) pseudosubstrate site RRGAI. In betaTC3 insulinoma cells, PDE7A1 co-localizes with PKA II in the Golgi-centrosome region. The roles PDE7A1 and its regulatory region play in cAMP signaling were examined by studying interactions with PKA subunits. PDE7A1 associates with the dissociated C subunit of PKA (C), but does not bind tetrameric PKA holoenzyme. High affinity binding of C by PDE7A1 inhibits kinase activity in vitro (IC50 = 0.5 nm). The domain containing PKA pseudosubstrate sites at the N terminus of PDE7A1 mediates complex formation with C. The PDE7A1 N-terminal repeat region inhibits C activity in CHO-K1 cells and also suppresses C dependent, cAMP-independent, physiological responses in yeast. Thus, PDE7A1 possesses a non-catalytic activity that can contribute to the termination of cAMP signals via direct inhibition of C. This study identifies a novel inhibitor of PKA and a non-catalytic affect of a cyclic nucleotide phosphodiesterase.     

Seven Dictyostelium discoideum phosphodiesterases degrade three pools of cAMP and cGMP

The Dictyostelium discoideum genome uncovers seven cyclic nucleotide PDEs (phosphodiesterases), of which six have been characterized previously and the seventh is characterized in the present paper. Three enzymes belong to the ubiquitous class I PDEs, common in all eukaryotes, whereas four enzymes belong to the rare class II PDEs that are present in bacteria and lower eukaryotes. Since all D. discoideum PDEs are now characterized we have calculated the contribution of each enzyme in the degradation of the three important pools of cyclic nucleotides: (i) extracellular cAMP that induces chemotaxis during aggregation and differentiation in slugs; (ii) intracellular cAMP that mediates development; and (iii) intracellular cGMP that mediates chemotaxis. It appears that each cyclic nucleotide pool is degraded by a combination of enzymes that have different affinities, allowing a broad range of substrate concentrations to be degraded with first-order kinetics. Extracellular cAMP is degraded predominantly by the class II high-affinity enzyme DdPDE1 and its close homologue DdPDE7, and in the multicellular stage also by the low-affinity transmembrane class I enzyme DdPDE4. Intracellular cAMP is degraded by the DdPDE2, a class I enzyme regulated by histidine kinase/phospho-relay, and by the cAMP-/cGMP-stimulated class II DdPDE6. Finally, basal intracellular cGMP is degraded predominantly by the high-affinity class I DdPDE3, while the elevated cGMP levels that arise after receptor stimulation are degraded predominantly by a cGMP-stimulated cGMP-specific class II DdPDE5. The analysis shows that the combination of enzymes is tuned to keep the concentration and lifetime of the substrate within a functional range.     

Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei

The identity and location of nuclear cyclic nucleotide phosphodiesterases (PDE) has yet to be ascertained. Intact cardiac nuclei and subnuclear fractions from ovine hearts were isolated to determine cAMP-specific PDE activity which was 3-fold greater than that of cGMP PDE, the latter being insensitive to Ca-calmodulin and zaprinast. Specific hydrolytic activities of the cardiac nuclear envelopes (NE) were similar to those measured in the corresponding intact nuclei, thus suggesting that most PDE activity is associated with the nuclear membrane. Moreover, the main hydrolytic activities in cardiac nuclei were attributed to PDE4 (56%) and PDE3 (44%). The pharmacological sensitivity of each isoform in terms of IC(50), K(m) and K(i) values was typical of previously characterized cardiac PDE 3 and 4 isoforms. PDE2 (cGMP-stimulated PDE) represented a minor component (8-9%) of total hydrolytic activity. Solubilization of nuclear envelopes and HPLC separation also yielded rolipram-sensitive PDE activities. Upon 1% Triton X-100 extractions, the presence of PDE3 and PDE4 was revealed in a low speed, nucleopore complex-enriched, P1 pellet. In addition, Western blot analysis demonstrated the presence of PDE4B and PDE4D subtypes in the nuclei as well as enrichment in NE. However, in the same preparations, the presence of PDE4A could not be ascertained. Altogether, these results suggest an intrinsic and predominant association of these nuclear PDEs with the NE and much likely with nucleopore complexes.     

Effects of fatty acids on activity of cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

Effects of fatty acids, prostaglandins, and phospholipids on the activity of purified cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver were investigated. Prostaglandins A2, E1, E2, F1 alpha, and F2 alpha, thromboxane B2, and most phospholipids were without effect; lysophosphatidylcholine was a potent inhibitor. Several saturated fatty acids (carbon chain length 14-24), at concentrations up to 1 mM, had little or no effect on hydrolysis of 0.5 microM [3H]cGMP or 0.5 microM [3H]cAMP with or without 1 microM cGMP. In general, unsaturated fatty acids were inhibitory, except for myristoleic and palmitoleic acids which increased hydrolysis of 0.5 microM [3H]cAMP. The extent of inhibition by cis-isomers correlated with the number of double bonds. Increasing concentrations of palmitoleic acid from 10 to 100 microM increased hydrolysis of [3H]cAMP with maximal activation (60%) at 100 microM; higher concentrations were inhibitory. Palmitoleic acid inhibited cGMP hydrolysis and cGMP-stimulated cAMP hydrolysis with IC50 values of 110 and 75 microM, respectively. Inhibitory effects of palmitoleic acid were completely or partially prevented by equimolar alpha-tocopherol. Palmitelaidic acid, the trans isomer, had only slightly inhibitory effects. The effects of palmitoleic acid (100 microM) were dependent on substrate concentration. Activation was maximal with 1 microM [3H]cAMP and was reduced with increasing substrate; with greater than 10 microM cAMP, palmitoleic had no effect. Inhibition of cGMP hydrolysis was maximal at 2.5 microM cGMP and was reduced with increasing cGMP; at greater than 100 microM cGMP palmitoleic acid increased hydrolysis slightly. Palmitoleic acid did not affect apparent Km or Vmax for cAMP hydrolysis, but increased the apparent Km (from 17 to 60 microM) and Vmax for cGMP hydrolysis with little or no effect on the Hill coefficient for either substrate. These results suggest that certain hydrophobic domains play an important role in modifying the catalytic specificity of the cGMP-stimulated phosphodiesterase for cAMP and cGMP.     

Involvement of cAMP-dependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation

We report the results of experiments which support the hypothesis that, in mouse oocytes, a decrease in intraoocyte cyclic AMP (cAMP) initiates meiotic maturation; oocytes microinjected with cyclic nucleotide phosphodiesterase (PDE) underwent germinal vesicle breakdown (GVBD) in the presence of 3-isobutyl-1-methylxanthine (IBMX), which inhibited GVBD both in oocytes not injected with PDE and in oocytes injected with heat-inactivated PDE. Cyclic AMP-dependent protein kinase (PK) has been proposed to mediate maintenance of meiotic arrest by cAMP. In support of this hypothesis is the observation that 2'-deoxy cAMP, which does not activate PK, did not maintain meiotic arrest as did cAMP; this result was obtained both by microinjection of these compounds and by incubating oocytes in the presence of their membrane-permeable N6-monobutyryl derivatives. Furthermore, microinjection into oocytes of the heat-stable inhibitor of PK, PKI, induced GVBD in the presence of either dibutyryl cAMP (dbcAMP) or IBMX. Meiotic arrest was maintained in the absence of dbcAMP or IBMX, however, by microinjected catalytic subunit of PK, but not by catalytic subunit coinjected with PKI. In addition, specific changes in oocyte phosphoproteins that preceded resumption of meiosis were induced, in the presence of dbcAMP, by microinjected PKI; these changes were also tightly coupled with commitment of oocytes to resume meiosis. These results are discussed in terms of our model for regulation of meiotic arrest and maturation.     

Effects of pH on allosteric and catalytic properties of the guanosine cyclic 3',5'-phosphate stimulated cyclic nucleotide phosphodiesterase from calf liver

We have investigated effects of pH on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver. In the "activated" state, i.e., with 0.5 microM [3H]cAMP plus 1 microM cGMP or at saturating substrate concentrations (250 microM [3H]cAMP or [3H]cGMP), hydrolysis was maximal at pH 7.5-8.0 in assays of different pH. Hydrolysis of concentrations of substrate not sufficient to saturate regulatory sites and below the apparent Michaelis constant (Kmapp), i.e., 0.5 microM [3H]cAMP or 0.01 microM [3H]cGMP, was maximal at pH 9.5. Although hydrolysis of 0.5 microM [3H]cAMP increased with pH from 7.5 to 9.5, cGMP stimulation of cAMP hydrolysis decreased. As pH increased or decreased from 7.5, Hill coefficients (napp) and Vmax for cAMP decreased. Thus, assay pH affects both catalytic (Vmax) and allosteric (napp) properties. Enzyme was therefore incubated for 5 min at 30 degrees C in the presence of MgCl2 at various pHs before assay at pH 7.5. Prior exposure to different pHs from pH 6.5 to 10.0 did not alter the Vmax or cGMP-stimulated activity (assayed at pH 7.5). Incubation at high (9.0-10.0) pH did, in assays at pH 7.5, markedly increase hydrolysis of 0.5 microM [3H]cAMP and reduce Kmapp and napp. After incubation at pH 10, hydrolysis of 0.5 microM [3H]cAMP was maximally increased and was similar in the presence or absence of cGMP. Thus, after incubation at high pH, the phosphodiesterase acquires characteristics of the cGMP-stimulated form. Activation at high pH occurs at 30 degrees C but not 5 degrees C, requires MgCl2, and is prevented but not reversed by ethylenediaminetetraacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)