Purification and characterization of a human platelet cyclic nucleotide phosphodiesterase

A cyclic nucleotide phosphodiesterase was extensively purified from the 100000g supernatant fraction of human platelets. The purification was 2500-3000-fold with 30% recovery of activity. The enzyme was isolated by DEAE-cellulose chromatography followed by adsorption to blue dextran-Sepharose and elution with cAMP. The protein has a molecular weight of 140 000 as determined by gel filtration. On NaDodSO4-containing polyacrylamide gels the major band is at 61 000 daltons, suggesting that the enzyme may exist as a dimer in solution under nondenaturing conditions. The enzyme requires Mg2+ or Mn2+ for activity. The calcium binding protein calmodulin does not stimulate hydrolysis of cAMP by this enzyme. The purified enzyme hydrolyzes both cAMP and cGMP with normal Michaelis-Menten kinetics with Km values of 0.18 microM and 0.02 microM, respectively. The hydrolysis of cGMP, however, is only one-tenth as rapid as the hydrolysis of cAMP. Cyclic GMP does not stimulate cAMP hydrolysis but instead is a potent competitive inhibitor of cAMP hydrolysis. The enzyme is also competitively inhibited by the phosphodiesterase inhibitors papaverine, 3-isobutyl-l-methylxanthine, and dipyridamole. The enzyme did not cross-react with an antibody raised to a cAMP phosphodiesterase isolated from dog kidney, indicating that the enzymes are not immunologically related. The inhibition of cAMP hydrolysis by cGMP suggests a possible regulatory link between these two cyclic nucleotides. One of the roles of cGMP in platelets may be to potentiate increases in intracellular cAMP by inhibiting the hydrolysis of cAMP by this enzyme.     

Purification and properties of cyclic nucleotide phosphodiesterase from uterine tissue

Cyclic nucleotide phosphodiesterase from calf myometrium has been purified to a homogeneous state for the first time, as can be evidenced from polyacrylamide gel electrophoresis data. The purification procedure included ion-exchange chromatography on DEAE-cellulose, high pressure liquid chromatography on TSK 545 DEAE and gel filtration through Toyopearl HW-55. The molecular mass of the enzyme as determined by gel filtration and polyacrylamide gel electrophoresis is 110 kD. The purified enzyme hydrolyzes cAMP and cGMP with Km = 30 microM and 18 microM, respectively.     

Purification of a cyclic nucleotide phosphodiesterase from bovine brain using blue dextran-Sepharose chromatography.

The soluble high Km form of cyclic nucleotide phosphodiesterase (EC 3.4.1.17) was purified over 2000-fold from bovine brain homogenates principally using blue dextran-Sepharose chromatography. The purified protein has a specific enzymic activity of 167 units/mg and appears homogeneous when examined by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 1.26 +/- 0.05 x 10(5) consisting of two apparently identical polypeptide chains. Kinetic measurements indicate that the substrates cyclic GMP and cyclic AMP each have a single Km value, 9 +/- 1 micron and 150 +/- 50 micron, respectively, that the two cyclic nucleotides compete for the same catalytic site, that the blue dye of blue dextran-Sepharose is a competitive inhibitor for the cyclic nucleotides, and that the Vmax with cyclic AMP as substrate is about an order of magnitude larger than that for cyclic GMP. Bovine brain calmodulin stimulates the catalytic rate of the purified enzyme in the presence of Ca2+ by increasing the Vmax associated with each cyclic nucleotide substrate.     

The cyclic nucleotide phosphodiesterase inhibitory protein of Dictyostelium discoideum. Purification and characterization

We have purified the glycoprotein inhibitor of the extracellular cyclic nucleotide phosphodiesterase of Dictyostelium discoideum to apparent homogeneity. The inhibitor has a molecular weight of 47,000 measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The interaction of the inhibitor and the cyclic nucleotide phosphodiesterase occurs with 1:1 stoichiometry and with a dissociation constant of about 10(-10) M. Periodate oxidation of the inhibitor or of the enzyme destroys concanavalin A binding ability but does not affect the formation of the enzyme-inhibitor complex. Inhibitor is not produced by cells during logarithmic growth but appears in quantity during stationary phase and after transfer from growth medium to phosphate buffer.     

Purification and characterization of cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Effects of divalent cations on activity

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase purified greater than 13,000-fold to apparent homogeneity from calf liver exhibited a single protein band (Mr approximately 102,000) on polyacrylamide gel electrophoresis under denaturing conditions. Enzyme activity comigrated with the single protein peak on analytical polyacrylamide gel electrophoresis, sucrose density gradient centrifugation, and gel filtration. From the sedimentation coefficient of 6.9 S and Stokes radius of 67 A, an Mr of 201,000 and frictional ratio (f/fo) of 1.7 were calculated, suggesting that the native enzyme is a nonspherical dimer of similar, if not identical, peptides. The effectiveness of Mg2+, Mn2+, and Co2+ in supporting catalytic activity depended on the concentration of cGMP and cAMP present as substrate or effector. Over a wide range of substrate concentrations, optimal concentrations for Mg2+, Mn2+, and Co2+ were about 10, 1, and 0.2 mM, respectively. At concentrations higher than optimal, Mg2+ inhibited activity somewhat; inhibition by Co2+ (and in some instances by Mn2+) was virtually complete. At low substrate concentrations, activity with optimal Mn2+ was equal to or greater than that with Co2+ and always greater than that with Mg2+. With greater than or equal to 0.5 microM cGMP or 20 to 300 microM cAMP and for cAMP-stimulated cGMP or cGMP-stimulated cAMP hydrolysis, activity with Mg2+ greater than Mn2+ greater than Co2+. In the presence of Mg2+, the purified enzyme hydrolyzed cGMP and cAMP with kinetics suggestive of positive cooperativity. Apparent Km values were 15 and 33 microM, and maximal velocities were 200 and 170 mumol/min/mg of protein, respectively. Substitution of Mn2+ for Mg2+ increased apparent Km and reduced Vmax for cGMP with little effect on Km or Vmax for cAMP. Co2+ increased Km and reduced Vmax for both. cGMP stimulated cAMP hydrolysis approximately 32-fold in the presence of Mg2+, much less with Mn2+ or Co2+. In the presence of Mg2+, Mn2+ and Co2+ at concentrations that increased activity when present singly inhibited cGMP-stimulated cAMP hydrolysis. It appears that divalent cations as well as cyclic nucleotides affect cooperative interactions of this enzyme. Whereas Co2+ effects were observed in the presence of either cyclic nucleotide, Mn2+ effects were especially prominent when cGMP was present (either as substrate or effector).     

Ca2+-dependent cyclic nucleotide phosphodiesterase from rabbit lung.

Studies are presented which demonstrate that rabbit lung contains both Ca²⁺-activated cyclic nucleotide phosphodiesterase and calmodulin activity. The Ca²⁺-activatable cyclic nucleotide phosphodiesterase is different from the common type in that it contains tightly bound calmodulin. The bound calmodulin is not dissociated from the enzyme even in very low concentrations of Ca²⁺ after DEAE-cellulose and Sephadex G-200 column chromatography.     

Ca2+-calmodulin-activated cyclic nucleotide phosphodiesterase from the rabbit myometrium

Two forms of soluble phosphodiesterase of cyclic nucleotides separating by DEAE-cellulose ion-exchange chromatography and not only differing in physicochemical and catalytic parameters but also differently regulated by calmodulin are found in the doe myometrium. Calmodulin with 10(-7)-10(-5) M concentrations of Ca2+ promotes the two-fold activation of the 3':5'-AMP (but not of 3':5'-GMP) hydrolysis by the first form of phosphodiesterase. Trifluoperazine (10 microM) lowers the activating action of calmodulin. The second form of soluble phosphodiesterase is not sensitive to the action of both calmodulin and Ca2+. 3':5'-GMP (10 microM) inhibits the 3':5'-AMP hydrolysis by the first form of phosphodiesterase; calmodulin exerts no effect on this process. The data obtained testify to the possible participation of Ca2+ and calmodulin in Ca2+-calmodulin-dependent phosphodiesterase regulation of the content of cyclic nucleotides (3':5'-AMP, in particular) in the doe myometrium.     

Purification and characterization of bovine brain calmodulin-dependent protein kinase II. The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme

Bovine brain contains two calmodulin-dependent phosphodiesterase kinases which are separated on Sephacryl S-300 column. One of these kinases has been purified to homogeneity and shown to belong to the calmodulin-dependent protein kinase II family. Phosphorylation of the 63 kDa phosphodiesterase by this purified protein kinase results in the incorporation of 1.0 mol phosphate per mol subunit and an accompanying increase in Ca²⁺ concentrations required for the phosphodiesterase activation by calmodulin. The protein kinase undergoes autophosphorylation to incorporate 1.0 mol phosphate per mol of subunit of the enzyme and the autophosphorylated enzyme is active, independent of the presence of Ca²⁺. The autophosphorylation reaction as well as the protein kinase reaction are rendered Ca²⁺ independent in less than 15 seconds when approximately one mol phosphate per mol protein kinase is incorporated. The result suggests that activation of phosphodiesterase phosphorylation reaction may occur prior to the activation of phosphodiesterase and phosphatase during a cell Ca²⁺ flux via the protein kinase autophosphorylation mechanism.Abbreviations SDS sodium dodecyl sulfate - EGTA ethylene glycol bis (-aminoethyl ether) - N,N,N,N tetra acetic acid - EDTA ethylenediamine-tetraacetic acid - cAMP cyclic adenosine 35 monophosphate This work is supported by grants from the Medical Research Council of Canada (JHW), the Heart and Stroke Foundation of Alberta (JHW and RKS) and the Heart and Stroke Foundation of Saskatchewan (RKS)     

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.     

Purification and characterization of a pyridine nucleotide glycohydrolase from rabbit spleen

A particulate NMN glycohydrolase of rabbit spleen was solubilized with Triton X100 and purified approximately 100-fold. The enzyme was shown to have a pH maximum of 6.5, a Km of 0.25 mM, a Vmax of 5.3 mumol/min/mg protein, an activation energy of 7.9 kcal/mol, and a molecular weight of approximately 400,000. Both of the purified and the particulate enzymes exhibited identical catalytic properties with respect to substrate specificity, activation energy, pH profile and exchange reaction with nicotinic acid, except that the purified enzyme was highly activated with Triton X100 as compared with the particulate enzyme; it appears that the purified enzyme possesses the same catalytic properties as the enzyme present in the tissue and that solubilization does not significantly alter the native protein. In addition to catalytic activity with NMN, the rabbit spleen enzyme catalyzed an irreversible hydrolysis with NAD and NADP, exhibiting catalyzing activity ratios of NMN:NAD:NADP = 1.00:1.45:0.44 and Vmax/Km ratios of 1.00:1.7:2.3, respectively. These ratios of activity remained constant throughout purification of the enzyme and no separation of these activities was detected. Mutually competitive inhibition of the enzyme with Ki values similar to Km, and identical rates of thermal denaturation of the enzyme and activity-pH profiles with NMN or NAD indicated the hydrolysis of the C-N glycosidic linkage of the pyridine nucleotides to be catalyzed by the same enzyme. The enzyme was less specific for the purine structure of the substrate dinucleotides but was stereospecific for the glycosidic linkage cleaved. Nicotinamide riboside, the nicotinic acid analogs and the reduced forms were not hydrolyzed. A linear noncompetitive inhibition of NMN hydrolysis with nicotinamide indicated an ordered Uni-Bi mechanism in which nicotinamide was the first product released from the enzyme. A property that the rabbit spleen enzyme appears to share with other NAD glycohydrolases is the transglycosidation reaction. The ratio of transglycosidation reaction vs. hydrolysis catalyzed by the enzyme in the presence of NMN and nicotinic acid indicated that the enzyme could function as a primary transglycosidase rather than a hydrolytic enzyme in vivo.     

Purification and characterization of human lung calmodulin-independent cyclic AMP phosphodiesterase

A high-affinity calmodulin-independent cyclic AMP phosphodiesterase was purified to homogeneity from human lung tissue. This enzyme has a molecular weight of 60,000, a sedimentation coefficient of 3.2–3.4 S, and an isoelectric pH of 4.6–4.8. Neither Ca²⁺ nor calmodulin (in the presence or absence of added Ca²⁺) stimulates the enzymatic activity. This enzyme appears to be very similar to that described previously from dog kidney (W. J. Thompson, P. M. Epstein, and S. J. Strada, (1979) Biochemistry18, 5228–5237). Hydrolysis of cyclic AMP is greatly enhanced by Mg²⁺ (25–30× at 10 mm Mg²⁺) and Mn²⁺ (20× at 10 mm Mn²⁺). Zn²⁺, Cu²⁺, and Co²⁺ are ineffective at these concentrations. Cyclic AMP is the exclusive substrate with a K_m of 0.7–0.8 μm. The I₅₀ of cyclic GMP is 1 mm using 1 μm cyclic AMP as substrate. In contrast, aminophylline, MIX, and SQ 20009 have I₅₀s of 0.28, 0.021, and 0.001 mm, respectively). The purified enzyme is susceptible to temperature inactivation and protease degradation. Significant (10%) inhibition is seen at 37 °C for 20 min. Trypsin, at 0.1 μg/ml, destroys 50% of the activity in 30 min at 25 °C. Our observations concerning its lability to temperature and proteases coupled with its lack of response to calmodulin suggest this enzyme is a basic catalytic subunit of other cyclic AMP phosphodiesterases present within human lung tissue.     

Two forms of cyclic nucleotide phosphodiesterase and Ca-dependent protein regulator from rabbit skeletal muscles]

In rabbit skeletal muscle extracts the activity of phosphodiesterase practically insensitive to the increase of Ca2+ concentration from 10(-8) M up to 10(-5) M. The Ca2+-dependent protein regulator is separated from phosphodiesterase at the stage of isolation and purification. The activity of phosphodiesterase devoid of the protein regulator is inhibited by Ca2+ (10(-5)--10(-3) M). An addition of Ca2+-dependent regulator protects the enzyme against inhibition by Ca2+. The Km values for 3',5'-AMP (5 mkM) and 3',5'-GMP (13 mkM) appear to be close; however, the maximal hydrolysis rates for these nucleotides differ considerably (14,0 and 0,25--0,50 nmoles/min/mg of protein). The hydrolysis of 3',5'-AMP is increased 1,6--3,2-fold under the effect of 3',5'-GMP and that of 3',5'-GMP is increased 1,8--2,7-fold under the effect of 3',5'-AMP. Using ion-exchange chromatography it was shown that only 1% of the total activity of skeletal muscle phosphodieterase belongs to the phosphodiesterase sensitive to the activating effect of Ca2+-dependent regulator the activity of this enzymic form is increased 4--5 fold. The Ca2+-dependent regulator of skeletal muscles is inactivated under the effects of trypsin and during gel-filtration is eluted together with the Ca2+-dependent regulator from the heart. The amount of Ca2+-dependent regulator in skeletal muscles is 30 times as low as that in brain and 3 times as low as that in the heart of the rabbit.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain identification of phospholipids as calcium-independent activators.

Phosphatidyl inositol and lysophosphatidyl choline have been identified as activators of a partially purified brain cyclic nucleotide phosphodiesterase previously shown to be regulated in vitro by Ca²⁺ and a Ca²⁺-binding protein. Microgram quantities of either phospholipid produced a linear, immediate and reversible activation of the enzyme in the absence of Ca²⁺ and the Ca²⁺-dependent regulator (CDR). Fatty acids were also found to activate the phosphodiesterase to varying degrees, with oleic acid being the most effective. Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and lysophosphatidyl ethanolamine were not effective as activators. Only sodium dodecyl sulfate, of a variety of nonionic, cationic, and anionic detergents tested, activated the phosphodiesterase. Sodium dodecyl sulfate produced a modest degree of activation over a narrow concentration range, followed by enzyme denaturation at higher concentrations.The interaction of the phosphodiesterase with the phospholipid activators has been compared to its interaction with the Ca²⁺·CDR complex. Both Ca²⁺·CDR and lysophosphatidyl choline decreased the thermal stability of the enzyme to a similar extent. The apparent K_m of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 30 μm with guanosine-3′,5′-monophosphate (cGMP) as substrate and 1 mm with adenosine-3′,5′-monophosphate (cAMP) as substrate. With increasing lysophosphatidyl choline concentration, the apparent K_m for each nucleotide remained unchanged while the V increased. The apparent K_d for Mg²⁺ of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 3 μm and was unaffected by lysophosphatidyl choline concentration. Activation of the phosphodiesterase by lysophosphatidyl choline was characterized by a high degree of positive cooperativity, exhibiting a Hill coefficient of 3.8. Fluphenazine was a competitive inhibitor of both Ca²⁺·CDR and lysophosphatidyl choline activation of the enzyme.     

Isolation and partial characterization of a cyclic GMP-dependent cyclic GMP-specific phosphodiesterase from Dictyostelium discoideum

The cellular slime mold, Dictyostelium discoideum, contains at least two classes of phosphodiesterase activity. One class of enzymes hydrolyses cyclic AMP (cAMP) and cyclic GMP (cGMP) with approximately equal rates. Another enzyme, which is less than 5% of the total activity, specifically hydrolyses cGMP. The cGMP-specific enzyme does not bind to a Con A-Sepharose column, while all the cAMP-hydrolyzing activities are retarded by this column. The cGMP-specific enzyme is activated by low cGMP concentrations (10^?8-10^?6 M); the enzyme has normal Michaelis-Menten kinetics at high substrate concentrations with a K_m of about 3–6 μM. The cGMP-binding sites for activation and for catalysis show different cyclic nucleotide specificity, but they are probably located on one protein with a molecular weight of about 70 000. The enzyme is stable only under specific conditions, and the activation property of the enzyme is lost relatively easy. Irreversible modifications occur at temperatures below 0° and above 30°C, and at pH below 6.0. Several other conditions such as high ion concentrations, temperatures just above 0°C and pH above 8.0 lead to reversibel modifications of enzyme activity.     

Biochemical and pharmacological characterization of cyclic nucleotide phosphodiesterase in airway epithelium

According to their respective elution order, specificity for cAMP and cGMP, their sensitivity to calmodulin, and their modulation by cGMP and rolipram, four cyclic nucleotide phosphodiesterases (PDE) were separated from the cytosol: PDE I (calmodulin-sensitive), PDE II (stimulated by cGMP, PDE IV (cGMP specific-PDE and inhibited by rolipram) and PDE V (cGMP specific). PDE IV (K_m=1.4 M) was competitively inhibited rolipram (K_i=1.2 M) whereas PDE V (K_m=0.83 M) was competitively inhibited by zaprinast in the molar range (K_i=0.12 M). Moreover the microsomal fraction contained three PDE isoforms: PDE II, PDE III (inhibited by cGMP or indolidan) and PDE IV. These results show that cAMP degradation in cytosolic and membrane fractions is modulated by cGMP and selectively inhibited by rolipram and, in addition, by indolidan in membrane fractions. (Mol Cell Biochem140: 171–175, 1994)