Cyclic nucleotide phosphodiesterase in rat neostriatum: multiple isoelectric forms with similar kinetic properties

Separation of multiple forms of cyclic nucleotide phosphodiesterase from the soluble supernatant fraction of rat neostriatum by isoelectric focusing yielded five separate peaks of cyclic nucleotide hydrolysing activity. Each separated enzyme form displayed a complex kinetic pattern for the hydrolysis of both cyclic AMP and cyclic GMP, and there were two apparent Km's for each nucleotide. At 1 microM substrate concentration, four enzyme forms exhibited higher activity with cyclic AMP than with cyclic GMP, while one form yielded higher activity with cyclic GMP than with cyclic AMP. Cyclic AMP and cyclic GMP were both capable of almost complete inhibition of the hydrolysis of the other nucleotide in all the peaks separated by isoelectric focusing; the IC50's for this interaction correlated well with the relative rates of hydrolysis of each nucleotide in each peak. The ratio of activity at 1 microM substrate concentration for the five enzyme forms separated by isoelectric focusing was 10:10:5:15:1 for cyclic AMP hydrolysis; and 6:6:4:8:2 for cyclic GMP hydrolysis; and the isoelectric points of the five peaks were 4.3, 4.45, 4.7, 4.85, and 5.5, respectively. Known phosphodiesterase inhibitors did not preferentially inhibit any of the separated forms of activity for either cyclic AMP or cyclic GMP hydrolysis, at either high (100 microM) or low (1 microM) substrate concentrations. Preliminary examination of the subcellular distribution of the different forms of enzyme activity indicated a different degree of attachment of the various forms to particulate tissue components. Isoelectric focusing of the soluble supernatant of rat cerebellum gave rise to a slightly different pattern of isoelectric forms from the neostriatum, indicating a different cellular distribution of the isoelectric forms of PDE in rat brain. Polyacrylamide disc gel electrophoresis of the soluble supernatant of rat neostriatum also generated a characteristic pattern of five separate peaks of cyclic nucleotide phosphodiesterase activity, each of which hydrolysed both cyclic AMP and cyclic GMP. Polyacrylamide gel electrophoresis of single enzyme forms previously separated by isoelectric focusing gave single peaks, with a marked correspondence between the enzyme forms produced by isoelectric focusing and those produced by gel electrophoresis, suggesting that both protein separation procedures were isolating the same enzyme forms. The results indicate the existence of multiple isoelectric forms of cyclic nucleotide phosphodiesterase in the soluble supernatant fraction of rat neostriatum, all of which exhibit similar properties. In this tissue a single kinetic form of this enzyme appears to exist displaying complex kinetic behaviour indicative of negative cooperativity and hydrolysing both cyclic AMP and cyclic GMP, with varying affinities.     

Isolation of an activator of multiple forms of cyclic nucleotides phosphodiesterase of rat cerebrum by isoelectric focusing.

An isoelectric focusing technique was used to isolate multiple forms of cyclic nucleotide phosphodiesterase from a 105 000 times g soluble supernatant fraction of sonicated rat cerebrum. These separated peaks of activity had iso-electric points of 5.1, 5.6, 6.0, 6.6, 8.0, and 9.0. The activities were not stimulated by an endogenous activator of the enzyme but were inhibited by EGTA treatment. However, activator-sensitive forms of the enzyme could be separated from brain if the preparation of rat cerebrum was dialyzed against an EGTA containing buffer prior to electrofocusing. The procedure was also used to isolate a column fraction that stimulated maximum velocities of cyclic AMP and cyclic GMP hydrolysis. This fraction was itself devoid of phosphodiesterase activity and had an isoelectric point of 4.7.     

Evidence for convertible forms of soluble uterine cyclic nucleotide phosphodiesterase

The cyclic nucleotide phosphodiesterase (3':5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) systems of many tissues show multiple physical and kinetic forms. In contrast, the soluble rat uterine phosphodiesterase exists as a single enzyme form with non-linear Lineweaver-Burk kinetics for cyclic AMP (app. Km of approx. 3 and 20 microM) and linear kinetics for cyclic GMP (app. Km of approx. 3 microM) since the two hydrolytic activities are not separated by a variety of techniques. In uterine cytosolic fractions, cyclic AMP is a non-competitive inhibitor of cyclic GMP hydrolysis (Ki approx. 32 microM). Also, cyclic GMP is a non-competitive inhibitor of cyclic AMP hydrolysis (Ki approx 16 microM) at low cyclic GMP/cyclic AMP substrate ratios. However, cyclic GMP acts as a competitive inhibitor of cyclic AMP phosphodiesterase (Ki approx 34 microM) at high cyclic GMP/cyclic AMP substrate ratios. When a single hydrolytic form of uterine phosphodiesterase, separated initially by DEAE anion-exchange chromatography, is treated with trypsin (0.5 microgram/ml for 2 min) and rechromatographed on DEAE-Sephacel, two major forms of phosphodiesterase are revealed. One form elutes at 0.3 M NaOAc- and displays anomalous kinetics for cyclic AMP hydrolysis (app. Km of 2 and 20 microM) and linear kinetics for cyclic GMP (app. Km approx. 5 microM), kinetic profiles which are similar to those of the uterine cytosolic preparations. A second form of phosphodiesterase elutes at 0.6 M NaOAc- and displays a higher apparent affinity for cyclic AMP (app. Km approx. 1.5 mu) without appreciable cyclic GMP hydrolytic activity. These data provide kinetic and structural evidence that uterine phosphodiesterase contains distinct catalytic sites for cyclic AMP and cyclic GMP. Moreover, they provide further documentation that the multiple forms of cyclic nucleotide phosphodiesterase in mammalian tissues may be conversions from a single enzyme species.     

Analysis of cyclic nucleotide phosphodiesterase by isoelectric focusing coupled to a specific activity stain

The procedure described allowed a convenient analysis of cyclic nucleotide phosphodiesterase. The different phosphodiesterase forms present in a crude cytosolic preparation from rat heart were separated by isoelectric focusing on a polyacrylamide gel plate. Phosphodiesterase activity bands were rendered evident by a specific staining method. They were then characterized by means of their substrate specificity and their sensitivity to selective phosphodiesterase inhibitors. The correspondence between the stain bands and the previously described activity peaks, obtained by a preparative technique and detected by radioisotopic enzyme assay, was also investigated.     

Analysis of cyclic nucleotide phosphodiesterase(s) by radioimmunoassay

A high-affinity form of cyclic AMP phosphodiesterase, purified to apparent homogeneity from dog kidney, was labeled with ¹²⁵I using a solid-state lactoperoxidaseglucose oxidase system and its purity confirmed by acrylamide gel electrophoresis and isoelectric focusing. Sheep anti-cyclic AMP phosphodiesterase immunoglobulin fraction was analyzed for ¹²⁵I-enzyme binding and covalently bound to agarose A 1.5m for isotopically labeled antigen displacement. Anti-phosphodiesterase antiserum was purified by Sepharose 4B-cAPDE affinity chromatography and used for a radioimmunoassay employing second-antibody precipitation. The specificity of the anti-cyclic AMP phosphodiesterase antibody was established by its use as a covalently bound affinity ligand for cyclic AMP phosphodiesterase purification and analysis of sodium dodecyl sulfate-gel extracts of partially purified and purified dog kidney supernatants. Radioimmunoassay using a monospecific antibody preparation demonstrated the similarity of high-affinity cyclic AMP phosphodiesterase forms of different tissues and species that had been separated by DEAE-cellulose chromatography. Various purified preparations of calmodulin, as well as brain calcineurin, did not cross-react in the high-affinity cyclic AMP phosphodiesterase radioimmunoassay. However, higher molecular weight cyclic GMP/lower affinity cyclic AMP phosphodiesterase enzyme forms, partially purified by anion-exchange chromatography, gel filtration, and Cibacron blue adsorption, were shown to cross-react in the high-affinity cAMP phosphodiesterase radioimmunoassay. These studies suggest immunological similarities between the major forms of this enzyme system and the possibility of higher molecular weight complexes containing both cyclic GMP and cyclic AMP hydrolytic sites.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

Cyclic adenosine 3':5'-monophosphate phosphodiesterase. Distinct forms in human lymphocytes and monocytes.

Adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase activity of normal human peripheral blood leukocyte suspensions containing 90% lymphocytes and 10% monocytes showed anomalous kinetic behavior indicative of multiple enzyme forms. Kinetic analyses of purified lymphocyte (99%) or monocyte preparations (95%) indicated that only one type of phosphodiesterase was present in each cell type. None of the preparations contained any detectable guanosine 3':5'-monophosphate (cyclic GMP) hydrolytic activity. The lymphocyte enzyme had an apparent Km congruent to 0.4 muM for cyclic AMP and Vmax congruent to 0.5 picomoles/min/10(6) cells. These kinetic parameters were confirmed by several cell purification techniques used alone and sequentially. Sedimentation velocity analyses indicated that the higher Km monocyte enzyme had a molecular weight near 45,000 and that the lower Km lymphocyte enzyme most likely had a molecular weight near 98,000. A variety of procedures led to a loss of the higher molecular weight, high affinity enzyme leaving only the enzyme of 45,000 daltons with a much lower substrate affinity. A long term, stable human lymphoblastoid cell line had cyclic AMP phosphodiesterase activity that was similar to the lymphocyte enzyme by both physical and kinetic criteria. Lymphocyte cyclic AMP phosphodiesterase appears to be a soluble enzyme whose pH and temperature optima and cationic requirements are similar to those of other mammalian phosphodiesterases. The distinct cyclic AMP phosphodiesterase forms of these cells may possibly represent the basic, active subunit of mammalian cyclic nucleotide phosphodiesterases. We hypothesize that the extremely high affinity cyclic AMP phosphodiesterase of normal lymphocytes plays an important role in the regulation of normal function in these cells, and also in the rapid proliferative responses characteristic of the stimulated lymphocyte.     

A comparison of the membrane-bound and extracellular cyclic AMP phosphodiesterases of Dictyostelium discoideum

The Dictyostelium discoideum membrane-bound and extracellular cyclic nucleotide phosphodiesterases (EC 3.1.4.17) shear several properties including the ability to react with a specific glycoprotein inhibitor and small inhibitory molecules. We have partialy purified the membrane-bound enzyme and compared its properties to those of the extracellular form. The kinetic properties of the two forms were similar except that, while associated with membrane particles, the membrane-bound form exhibited non-linear kinetics when assayed ove a broad substrate range. The isoelectric point of the membrane-bound phosphodiesterase was identical to that of the extracellular enzyme when isoelectrofocusing was done in the presence of 6 M urea. The molecular weights of membrane-bound and extracellular enzyme, determined by gel filtration, were the same following isoelectrofocusing in the presence of 6 M urea. When precipitated with an antiserum prepared against purified extracellular phosphodiesterase, the partially purified membrane-bound enzyme preparation was shown to contain a M_r 50 000 polypeptide comigrating with the extracellular enzyme during SDS polyacrylamide gel electrophoresis. When the iodinated extracellular enzyme and the iodinated M_r 50 000 polypeptide from membrane-bound enzyme were subjected to partial proteolytic digestion, similar profiles were obtained indicating extensive regions of homology.     

Characterization of particulate cyclic nucleotide phosphodiesterases of rat kidney.

Particulate cyclic nucleotide phosphodiesterases of rat kidney display some distinct kinetic and regulatory properties. Only a small portion (5–10%) of the total homogenate low K_m cyclic AMP phosphodiesterase activity (measured with concentrations of cyclic AMP less than l μm) is tightly associated with kidney membranes. Cyclic GMP phosphodiesterase activity (measured with 0.25–200 μm cyclic GMP) is readily detectable in these fractionated and washed membranes. Low concentrations of cyclic GMP stimulated the hydrolysis of cyclic AMP (K_a ～- 0.5 μM), an effect not noted in most other membrane systems. High concentrations of cyclic GMP (K_i ～- 450 μM) and cyclic AMP (K_i ～- 150 μM) inhibited the hydrolysis of each other noncompetitively. Solubilization of membrane bound activities by sonication or Sarkosyl L markedly alters enzyme kinetic properties and the responses to cyclic nucleotides and sulfhydryl reagents. Incubation of membrane fractions with dithiothreitol (5 mm) or storage of the membranes at 4 °C results in a change in extrapolated kinetic constants for cyclic AMP hydrolysis and an increase in the rate of denaturation at 45 °C. Our findings raise the possibility that regulation of membrane-bound cyclic nucleotide phosphodiesterase activity involves interactions with cyclic nucleotides themselves, as well as oxidation and reduction of disulfide bonds and membrane-enzyme interactions.     

Thermostable extracellular cyclic nucleotide phosphodiesterase of the Physarum polycephalum plasmodium

Cyclic nucleotide phosphodiesterase secreted by the Physarum polycephalum plasmodium was partially purified by ion-exchange chromatography on DEAE cellulose, ultrafiltration, and HPLC. The data obtained by gel filtration, HPLC, electrophoresis, and isoelectric focusing showed that the active enzyme in solution exists as a monomer of about 90 kDa with pI 3.6–4.0. The K _m values were 0.9 and 7.7 mM for cAMP and cGMP, respectively, whereas the maximal rates of hydrolysis of these nucleotides were virtually equal and reached several millimoles of hydrolyzed cyclic nucleotide per hour per milligram of enzyme. The partially purified enzyme was highly stable. It was not inactivated by heating at 100°C for 30 min. The enzyme remained active in the presence of 1% sodium dodecyl sulfate; however, it was completely inactivated under these conditions in the presence of β-mercaptoethanol.     

Isoelectric-focusing patterns of cyclic nucleotide phosphodiesterase from rat heart.

1. Isoelectric focusing on a flat gel bed of the rat heart cytosolic fraction resolved cyclic nucleotide phosphodiesterase activity into several forms, characterized by their substrate specificity, kinetic constants and dependence towards Ca2+ and calmodulin. A peak of pI 4.9 displayed 20 times more affinity for cyclic GMP than for cyclic AMP and was markedly inhibited by EGTA. A less substrate-specific form, only slightly sensitive to EGTA inhibition, focused at pH 5.45. Several overlapping peaks detected between pH 5.55 and pH6 specifically hydrolysed cyclic AMP, with non-Michaelian kinetics; these peaks were insensitive to Ca2+ chelation. 2. Isoelectric focusing did not dissociate enzyme-calmodulin complexes, as none of the resulting peaks was activatable by calmodulin plus Ca2+. 3. Some new information on rat cardiac phosphodiesterase is obtained with this technique, which is convenient for routine analytical studies of phosphodiesterase, as well as for preparative purposes.     

Cyclic nucleotide phosphodiesterase from a particulate fraction of rat heart. Solubilization and characterization of a single enzymatic form

Approximatively 2–8% of the cyclic nucleotide phosphodiesterase activity of a crude 1000 g supernatant from rat heart was associated with the washed 105,000 g pellet fraction. This activity exhibited biphasic Lineweaver-Burk plots over a large range of cyclic nucleotides concentrations. Concave-Bownward plots were obtained with cyclic AMP as the assay substrate, while cyclic GMP gave rise to concave-upward plots. Treatment of this particulate fraction by freezing and thawing and then with 2% Lubrol PX released the major part of phosphodiesterase activity into the supernatant (70 and 90% for cyclic AMP and cyclic GMP phosphodiesterase activities respectively). Isoelectric focusing of the solubilized enzyme revealed a single peak of phosphodiesterase activity. While the Lineweaver-Burk plots of cyclic AMP phosphodiesterase activity were not markedly modified by detergent treatment kinetic plots of cyclic GMP phosphodiesterase activity underwent a drastic transformation during the overall solubilization procedure. The substantial increase in the cyclic GMP rate of hydrolysis observed at low substrate level might explain the difference in the apparent yield of solubilization between cyclic AMP and cyclic GMP phosphodiesterase activities.     

Cyclic nucleotide phosphodiesterase from a particulate fraction of rat brain. Evidence for an activator deficient form.

A cyclic nucleotide phosphodiesterase from a particulate fraction of rat brain was partially purified after solubilization with a non-ionic detergent. Influence of divalent ions, of phosphodiesterase inhibitors and of the activator protein from different sources were tested. Determination of K_m-values shows two enzymes with different values, one at 7.3 μM and the other at 15 mM. Two distinct activity peaks were determined after resolution by isoelectric focusing. It was concluded that this particulate enzyme is regulated in a way opposite to that of the soluble enzyme and is independent from calcium and the activator protein.     

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).     

A partial characterization of the cyclic nucleotide phosphodiesterases of Drosophila melanogaster

The cyclic nucleotide phosphodiesterases in crude homogenate, soluble material, and particulate preparations of adult Drosophila melanogaster flies, hydrolyze cyclic AMP with nonlinear kinetics. Cyclic GMP is hydrolyzed by the phosphodiesterases in crude homogenate and soluble material with linear kinetics. Physical separation techniques of gel filtration, velocity sedimentation, and ion-exchange chromatography reveal that Drosophila soluble fraction contains two major forms of cyclic nucleotide phosphodiesterase. Form I hydrolyzes both cyclic AMP and cyclic GMP. Inhibition experiments suggest that the hydrolysis of both cyclic nucleotides by Form I occurs at a single active site. The K_m's for hydrolysis of both substrates are about 4 μm. This form has a molecular weight of about 168,000 as estimated by gel nitration. Form II cyclic nucleotide phosphodiesterase is specific for cyclic AMP as substrate. Gel filtration indicates that this form has a molecular weight of about 68,000. The K_m for cyclic AMP is about 2 μm.     

Characterization of soluble uterine cyclic nucleotide phosphodiesterase.

Soluble cyclic nucleotide phosphodiesterase of rat uterus displays distinct structural and regulatory properties. Like phosphodiesterases from many mammalian sources the soluble uterine enzyme system exhibits nonlinear Lineweaver--Burk kinetics with cyclic adenosine 3':5'-monophosphate (cAMP) as substrate (apparent Kms congruent to 3 and 20 micron) and linear kinetics with cyclic guanosine 3':5'-monophosphate (cGMP) as substrate (apparent Km congruent to 3 micron). Unlike most other mammalian phosphodiesterases, however, numerous separation procedures reveal only a single form of uterine phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP. A single form of the enzyme is observed upon sucrose gradient centrifugation (7.9 S), agarose gel filtration, and DEAE-cellulose chromatography at either pH 8.0 OR 6.0. Heat denaturation (50 degrees C) of soluble uterine phosphodiesterase causes the loss of both cAMP and cGMP hydrolytic activities at the same rate. Isoelectric focusing reveals major (pI = 5.2) and minor forms (pI = 5.8) of phosphodiesterase which both catalyze the hydrolysis of the two cyclic nucleotide substrates. In vivo administration of estradiol produces identical decreases in the activities of cAMP and cGMP phosphodiesterase. These results raise the possibility that the uterus contains a single form of soluble phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP.     

Aggregation states of cyclic nucleotide phosphodiesterase of murine thymocytes

In murine thymocytes cyclic nucleotide phosphodiesterase is represented by cAMP- and cGMP-specific forms. cAMP and cGMP phosphodiesterase activities showed anomalous kinetic behaviour indicative of 'low' and 'high' affinity enzyme forms. Sucrose density gradient centrifugation resolved only 'low' affinity forms of cAMP and cGMP phosphodiesterases. Gel filtration on Ultragel Aca 34 column showed that cAMP and cGMP phosphodiesterases are probably oligomeric enzymes. Storage of enzyme preparation at 4 degrees C for 24-48 h led to a decrease of higher molecular weight form and enhancement of cAMP and cGMP phosphodiesterase activities.     

Adenylate cyclase and cyclic nucleotide phosphodiesterases in the developing rat liver

A potential regulatory role for the cyclic nucleotides during liver morphogenesis will be better understood as the development of various components of the cyclic nucleotide system are characterized. Accordingly, adenylate cyclase response to glucagon and 5′-guanylimidodiphosphate (Gpp(NH)p) and the specific activities, cellular distributions, and kinetic constants (V and K_m) of the cyclic AMP and cyclic GMP phosphodiesterases were determined at variuos stages of rat liver development. These results show (1) a period of increasing sensitivity of rat liver adenylate cyclase to glucagon at a time when sensitivity to NaF and Gpp(NH)p remains unchanged, and (2) increased responsiveness to glucagon plus Gpp(NH)p which is dependent upon the degree of glucagon sensitivity. It is concluded that the guanul nucleotide regulatory site is a functional part of adenylate cyclase very early in liver development and that the development of glucagon sensitivity is more probably limited by the developmet of glucagon receptors. Two forms of each phosphodiesterase (high and low K_m) were found throughout, except that low K_m cyclic GMP phosphodiesterase could not be demonstrated in the embryo. No significant change with age was found for the K_m or V of any of the enzyme forms. The ratio of soluble: particulate cyclic AMP phosphodiesterase decreased with age, whereas no change in the ration for cyclic GMP phosphodiesterase was observed. Specific activities of each enzyme from were highest in the perinatal period and decreased with age. The changes in phosphodiesterase specific activities paralled changes in guanylate and adenylate cyclase activities, which argues against a selective regulatory role for phosphodiesterase in modulating cyclic nucleotide influences during liver morphogenesis.     

The adnosine 3',5'-monophosphate and guanosine 3',5'-monophosphate phosphodiesterases from human lung tissue.

Human lung tissue contains phosphodiesterase enzymes capable of hydrolyzing both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP). The cyclic AMP enzyme exhibits three distinct binding affinities for its substrate (apparent Km = 0.4μM, 3μM, and 40μM) while the cyclic GMP enzyme reveals only two affinities (Km = 5μM and 40μM). The pH optima for the cyclic AMP and cyclic GMP phosphodiesterase are similar (pH 7.6–7.8). Both are inhibited by known inhibitors of phosphodiesterase activity (aminophylline, caffeine, and 3-isobutyl-1-methylxanthine). The divalent cations Mg²⁺ and Mn²⁺ stimulate cyclic AMP phosphodiesterase activity (in the absence of Mg²⁺) while Ca²⁺, Ni²⁺, and Cu²⁺ inhibit the enzyme. Histamine and imidazole slightly stimulate cyclic AMP hydrolytic activity. Thus, human lung tissue does contain multiple forms of both the cyclic AMP and cyclic GMP phosphodiesterase which are influenced by a variety of effectors.     

Human blood platelet 3': 5'-cyclic nucleotide phosphodiesterase. Isolation of low-Km and high-Km phosphodiesterase.

Human blood platelet contained at least three kinetically distinct forms of 3': 5'-cyclic nucleotide phosphodiesterase (3': 5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) (F I, F II, and F III) which were clearly separated by DEAE-cellulose column chromatography. Although a few properties of the platelet phosphodiesterases such as their substrate affinities and DEAE-cellulose profile resembled somewhat those of the three 3': 5'-cyclic nucleotide phosphodiesterase in rat liver reported by Russell et al. [10], there were pronounced differences in some properties between the platelet and the liver enzymes: (1) the platelet enzymes hydrolyzed both cyclic nucleotides and lacked a highly specific cyclic guanosine 3': 5'-monophosphate (cyclic GMP) phosphodiesterase and (2) kinetic data of the platelet enzymes indicated that cyclic adenosine 3': 5'-monophosphate (cyclic AMP) and cyclic GMP interact with a single catalytic site on the enzyme. F I was a cyclic nucleotide phosphodiesterase with a high Km for cyclic AMP and a negatively cooperative low Km for cyclic GMP. F II hydrolyzed cyclic AMP and cyclic GMP about equally with a high Km for both substrates. F III was low Km phosphodiesterase which hydrolyzed cyclic AMP faster than cyclic GMP. Each cyclic nucleotide acted as a competitive inhibitor of the hydrolysis of the other nucleotide by these three fractions with Ki values similar to the Km values for each nucleotide suggesting that the hydrolysis of both cyclic AMP and cyclic GMP was catalyzed by a single catalytic site on the enzyme. However, cyclic GMP at low concentration (below 10 muM) was an activator of cyclic AMP hydrolysis by F I. Papaverine and EG 626 acted as competitive inhibitors of each fraction with virtually the same Ki value in both assays using either cyclic AMP or cyclic GMP as the substrate. The ratio of cyclic AMP hydrolysis to cyclic GMP hydrolysis by each fraction did not vary significantly after freezing/thawing or heat treatment. These facts also suggest that both nucleotides were hydrolyzed by the same catalytic site on the enzyme. The differences in apparent Ki values for inhibitors such as cyclic nucleotides, papaverine and EG 626 would indicate that three enzymes were different from each other. Centrifugation in a continuous sucrose gradient revealed sedimentation coefficients F I and II had 8.9 S and F III 4.6 S. The molecular weight of these forms, determined by gel filtration on a Sepharose 6B column, were approx. 240 000 (F I and II) and 180 000 (F III). F III was purified extensively (70-fold) from homogenate, with a recovery of approximately 7%.