A new method for separating cyclic AMP from 5′-AMP with application to the assay for cyclic AMP phosphodiesterase

A new method for assay of cyclic AMP phosphodiesterase (EC 3.1.4.17) has been developed based on the observation that a mixture of cyclic AMP and AMP can be resolved on a column of florisil (activated magnesium silicate) at pH 7.0. The cyclic nucleotide is retained by the silicate and the AMP which is not adsorbed is virtually quantitatively recovered. The adsorption of cyclic AMP by florisil is greatly influenced by the pH of the buffer but independent of its ionic strength. In the actual assay cyclic[³H]AMP is incubated with the enzyme source in the presence of Mg²⁺ and the reaction is stopped by the addition of CCl₃COOH (0.3 m). The mixture is then neutralized by dilution with 10 vol of 0.5 m sodium phosphate buffer, pH 7.0, and applied on a small (0.4 × 4.0-cm) florisil column equilibrated with the same buffer. The column is eluted with 3 vol of the buffer and the radioactivity of the eluate which contains only [³H]AMP is measured. The use of cyclic[³H]AMP of high specific activity in the assay allows a high degree of sensitivity while the addition of CCl₃COOH instantaneously terminates the reaction allowing for increased precision. The assay compares favorably in simplicity and speed with those currently employed for cyclic AMP phosphodiesterase.     

Identification and characterization of a Ca2+-calmodulin-sensitive cyclic nucleotide phosphodiesterase in a human lymphoblastoid cell line.

This study examines the pattern and regulatory properties of cyclic nucleotide phosphodiesterases in a human lymphoblastoid B-cell line (RPMI 8392) established from a patient with acute lymphocytic leukaemia. In this cell line, phosphodiesterase activity measured at 0.25 microM-cyclic AMP is approx. 7-fold greater than that in isolated human peripheral-blood lymphocytes, and 16% of the phosphodiesterase activity in RPMI 8392 cells is associated with particulate fractions. Phosphodiesterase activity in crude fractions of this cell line is reproducibly stimulated by about 60-80% by Ca2+-calmodulin. In the presence of 20 nM-calmodulin, half-maximal stimulation occurs at 0.7 microM-Ca2+. The cytosolic phosphodiesterase activity of RPMI 8392 cells is separated into two forms by DEAE-Sephacel chromatography. The first form is eluted at approx. 0.2 M-sodium acetate, catalyses the hydrolysis of both cyclic AMP and cyclic GMP, and is stimulated 3-fold by Ca2+-calmodulin. This form exhibits non-linear kinetics for cyclic AMP in the absence of calmodulin, with extrapolated Km values of 0.8 and 4 microM, and non-linear kinetics in the presence of calmodulin, with extrapolated Km values of 0.5 and 1 microM. The Vmax. values are increased approx. 3-fold by calmodulin. The second form is eluted at approx. 0.6 M-sodium acetate, is specific for cyclic AMP, and insensitive to stimulation by Ca2+-calmodulin. The Ca2+-calmodulin-sensitive phosphodiesterase from the DEAE-Sephacel column can be adsorbed to a calmodulin-Sepharose affinity column and eluted with EGTA. This enzymic activity can also be immunoprecipitated by a monoclonal antibody directed against a calmodulin-bovine heart phosphodiesterase complex. This study documents the existence of Ca2+-calmodulin-sensitive phosphodiesterase in a cultured lymphoblastoid cell line derived from a leukaemic patient.     

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.     

Purification and kinetic properties of two soluble forms of calmodulin-dependent cyclic nucleotide phosphodiesterase from rat pancreas.

The calmodulin-dependent cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase (EC 3.1.4.17) activity of rat pancreas was purified 280-fold by affinity chromatography on calmodulin-Sepharose 4B. It then accounted for 15% of the total cytosol cyclic GMP nucleotide phosphodiesterase activity, in the presence of Ca2+, and represented a minor component of proteins specifically adsorbed by the column. This activity was resolved on a DEAE-Sephacel column into two fractions, termed PI and PII, on the basis of their order of emergence. After this step, PI and PII were purified 5650- and 3700-fold respectively. The molecular weight of PI was 175 000 and that of PII was 116 000, by polyacrylamide-gradient-gel electrophoresis. Both forms of phosphodiesterase could hydrolyse cyclic AMP and cyclic GMP, although PII displayed a higher affinity toward cyclic GMP than toward cyclic AMP. PI and PII exhibited negative homotropic kinetics in the absence of calmodulin. Upon addition of calmodulin, both enzymes displayed Michaelis-Menten kinetics and a 5-9-fold increase in maximal velocity, at physiological concentrations of cyclic GMP and cyclic AMP. When a pancreatic extract freshly purified by affinity chromatography was immediately analysed by high-performance gel-permeation chromatography on a TSK gel G3000 SW column, PII represented as much as 78% of the eluted activity. This percentage decreased to 52% when the sample was stored at 0 degrees C for 20 h before analysis, suggesting that PII, possibly predominant in vivo, was converted into the heavier PI form upon storage.     

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).     

Characterization of a rat liver cyclic GMP-activated phosphodiesterase by chromatography on hexyl-agarose. Inhibition of phosphodiesterase activity by hexyl-agarose.

Chromatography on hexyl-agarose resolved a partially purified cyclic GMP-activated phosphodiesterase from rat liver into two peaks of activity: the first was eluted with 0.5 M-KCl and was cyclic AMP-specific. The second was tightly bound to hexyl-agarose and was not eluted with KCl (0--2.0 M), which enhanced the hydrophobic interactions of this form with the matrix. It was eluted with 0.5 M-Tris, hydrolysed cyclic AMP and cyclic GMP and was specifically activated by cyclic GMP. The cyclic GMP-activated phosphodiesterase was immobilized on hexyl-agarose. Enzyme activity, quantitatively bound to hexyl-agarose, was not released from the hydrophobic matrix in the presence of cyclic AMP or cyclic GMP, under our assay conditions. The immobilized form of the enzyme retained catalytic activity, was inhibited by 0.1 mM-cyclic AMP and was activated by micromolar concentrations of cyclic GMP to a lesser extent (7-fold) than the control, i.e. the enzyme mixed with unsubstituted agarose (15-fold). When the enzyme was immobilized, inhibition of cyclic AMP phosphodiesterase activity was only observed in the presence of cyclic GMP (at 3 microM); in its absence, activity remained unchanged. The kinetic behaviour of the immobilized enzyme is consistent with the hypothesis of a binding site distinct from the hydrolytic and activating sites.     

Multiple protein kinases from human lymphocytes. Identification enzymes phosphorylating exogenous histon and casein.

1. Cell-free lysates of human peripheral blood lymphocytes contained two casein kinase activities and two histone kinase activities, which could be separated by chromatography on DEAE-Sephadex. 2. Neither of the casein kinase activities were stimulated by cyclic AMP. The major activity was eluted from DEAE-Sephadex between 0.4 and 0.45M-KCl, had a molecular weight of approx. 130,000 (sucrose density gradients) and was stimulated by KCl (maximum 150mM). It also formed higher-molecular-weight aggregates when centrifuged in sucrose gradients containing 150mM-KCl. The minor activity was not retained by DEAE-Sephadex, had a molecular weight of approx. 50,000 and was not stimulated by KCl. 3. The major histone kinase activity was stimulated by cyclic AMP and was eluted from the DEAE-Sephadex column between 0.05 and 0.2M-KCl. The other activity was not stimulated by cyclic AMP and was insensitive to the rabbit muscle protein kinase inhibitor. 4. Evidence was obtained suggesting that the lymphocyte casein kinases were located primarily in the nuclei.     

Calmodulin-Like Activity Associated with Chromatin from Pea Buds

NAD kinase and cyclic AMP phosphodiesterase were activated bya factor prepared from pea chromatin. About 62% of the originalamount of the factor in the purified chromatin was recoveredin the reassociated chromatin. The NAD kinase- and cyclic AMP phosphodiesterase-activatingfactor was released from the chromatin by heat treatment withethylene glycol-bis(ß-aminoethyl ether)- N,N,N',N'-tetraaceticacid (EGTA) then adsorbed on an affinity gel of phenothiazineagarosederivatives in the presence of excess Ca²⁺ over EGTA, afterwhich it was eluted by a flush of EGTA. Activation of NAD kinaseand cyclic AMP phosphodiesterase by this factor depended onthe presence of Ca²⁺. The NAD kinase-activating factor and chromatin were coelutedwhen soluble chromatin was applied to a Bio-Gel A50 column.When chromatin was chromatographed on the same column afterdigestion by DNase I, the factor was eluted in association withthe digested products of the chromatin. The activation propertiesof this factor indicate that a calmodulin-like activity existsin association with pea chromatin. The activation curves of cyclic AMP phosphodiesterase with thepea bud factor and with bovine brain calmodulin were compared.The amount of the factor in the chromatin fraction that correspondedto authentic calmodulin was calculated as 5.7 µg per mgDNA. (Received August 6, 1982; Accepted February 17, 1983)     

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.     

Purification of rat heart cyclic nucleotide phosphodiesterase on column of immobilized phenylbutenolide inhibitor

Cyclic nucleotide phosphodiesterase was purified over 200-fold in a single step from the rat heart cytosolic fraction, using affinity chromatography on phenylbutenolide inhibitor immobilized to AH Sepharose. After elimination of the contaminating proteins by washing with the loading buffer and then with 0.4 M KCl buffer, without any loss in enzymatic activity, the cyclic nucleotide phosphodiesterase was eluted in good zields with a linear KCl gradient from 0.4 M to 1.8 M. Enzymatic activity determination performed with both cyclic AMP and cyclic GMP as substrate, either at low (0.25 μM) or at high (25 μM) concentration, pointed out the presence of several phosphodiesterase forms with different substrate specificities, in the elution profiles.     

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.     

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.     

Solubilization and characterization of hormone- responsive phosphodiesterase activity of rat fat cells.

Fat cells particulate phosphodiesterase activity can be solubilized in high yield (80--100%) in a buffer system (30 mM Tris - HCl, pH 8.0) containing non-ionic detergents (0.1% Brij 30, 1.0% Triton X-100), salt (3.0 mM MgSO4, 5.0 mM NaBr) and dithiothreitol (5.0 mM). Polyacrylamide gel electrophoresis of the solubilized enzyme activity indicated the presence of two bands of activities of different electrophoretic mobilities, both of which hydrolyzed cyclic AMP and cyclic GMP. The solubilized activity eluted from DEAE Bio-Gel columns as a somewhat broad profile with at least two peaks of activity. Activity against both cyclic AMP and cyclic GMP eluted in similar but not identical patterns. The solubilized enzyme and DEAE column eluates wxhibited low (less than 1 micronM) Michaelis constants for cyclic AMP and cyclic GMP. In addition, the increases in phosphodiesterase activity induced by incubation of intact fat cells with insulin or adrenocorticotropic hormone are maintained in the solubilized state.     

Characterization of a calmodulin-dependent high-affinity cyclic AMP and cyclic GMP phosphodiesterase from male mouse germ cells.

Two cyclic nucleotide phosphodiesterase activities were separated by ion-exchange chromatography of cytosol from male mouse germ cells. A form eluted at low salt concentration showed high affinity (Km congruent to 2 microM) and low affinity (Km congruent to 20 microM) for cyclic AMP, and high affinity (Km congruent to 3.5 microM) for cyclic GMP. A second form, eluted at high salt concentration, showed high affinity (Km congruent to 5 microM) for cyclic AMP and was similar to a phosphodiesterase activity described in rat germ cells. The present study was performed to characterize the first form, which represents most of the phosphodiesterase activity in mouse germ cells. The enzyme was sensitive to Ca2+ and calmodulin stimulation, which increased its activity 3-4-fold. Calmodulin stimulation depended on direct interaction of the activator with the enzyme, as indicated by the reversible changes in the chromatographic elution pattern in the presence of Ca2+, as well as by the increase in the sedimentation coefficient in the presence of calmodulin. Reciprocal inhibition kinetics between cyclic AMP and cyclic GMP for the calmodulin-dependent form demonstrated a non-competitive inhibition between the two substrates, suggesting the presence of separate catalytic sites. This is in agreement with kinetic parameters and different thermal stabilities of cyclic AMP- and cyclic GMP-hydrolysing activities. Furthermore, the relevant change in s value, depending on the absence or presence of Ca2+ and calmodulin, suggested that the enzyme is composed of subunits, which aggregate in the presence of the activator. A model for catalytic site composition and reciprocal interaction is also proposed.     

The specific interaction of Cibacron and related dyes with cyclic nucleotide phosphodiesterase and lactate dehydrogenase

1. Reactive Blue 2 (Cibacron Blue 3G-A) is a competitive inhibitor of bovine heart cyclic nucleotide phosphodiesterase (K(i) 0.3mum). The K(i) increases with increasing temperature, suggesting that hydrophobic interactions are not largely responsible for the binding of the dye. Another 25 sulphonated aromatic dyes are also competitive inhibitors of the cyclic nucleotide phosphodiesterase (K(i) values in the range of 0.06-13.6mum). 2. These dyes (covalently linked to Dextran 40) inhibit bovine heart cyclic nucleotide phosphodiesterase. Reactive Blue 2 (covalently linked to Dextran 40) is a competitive inhibitor of the phosphodiesterase (K(i) 0.4mum). 3. Bovine heart cyclic nucleotide phosphodiesterase is retained on a column of Reactive Blue 2-Sephacryl S-200 and can be eluted from the column by 3':5'-cyclic AMP. 4. A variety of the dyes (either free or covalently linked to Dextran 40) are competitive inhibitors of rabbit muscle lactate dehydrogenase. 5. The effectiveness of a wide range of structurally dissimilar dyes as competitive inhibitors of lactate dehydrogenase and cyclic nucleotide phosphodiesterase compromises proposals for the use of Reactive Blue 2 as a specific probe for the dinucleotide-binding structural domain present in many dehydrogenases and kinases. Detailed information of the various dyes used has been deposited as Supplementary Publication SUP 50089 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Activation of cyclic AMP phosphodiesterase by a new vitamin E derivative.

The effects of sodium alpha-tocopherol phosphate (TPNa), a new vitamin E derivative, on cyclic nucleotide phosphodiesterases from a soluble supernatant fraction of rat liver were investigated. TPNa produced a dose-dependent increase in cyclic AMP hydrolysis at a low substrate concentration (1 muM cyclic AMP), whereas the compound inhibited the hydrolytic activity at a high substrate level (100 muM cyclic AMP). Cyclic GMP phosphodiesterase activity was suppressed by TPNa regardless of the substrate concentration. The addition of TPNa did not change the apparent Km value (50 muM) of cyclic AMP phosphodiesterase at low substrate level (less than 5 muM). In contrast, at higher substrate concentration, the concave downward curve observed in a Lineweaver-Burk plot became straight in the presence of TPNa. Low concentrations of cyclic GMP, which are known to activate cyclic AMP hydrolysis, showed an additive effect on cyclic AMP phosphodiesterase only when a submaximal concentration of cyclic GMP was present in addition to TPNa. These and other data suggest that TPNa modifies cyclic AMP phosphodiesterase in all allosteric fashion.     

Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurosproa crassa

Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca²⁺-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine.     

A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates.

A method for the separation of cyclic AMP from adenosine and polyvalent adenine nucleotides is described. The method consists of the sequential elution of adenosine and cyclic AMP from a single column of acidic aluminum oxide (alumina) with dilute hydrochloric acid and ammonium acetate. Adenosine, adenine, xanthine, and hypoxanthine are rapidly eluted with the application of 0.005 N hydrochloric acid while cyclic AMP remains adsorbed to the alumina. A subsequent application of 0.1 M ammonium acetate elutes more than 90% of the cyclic AMP. Under these conditions, polyvalent nucleotides (AMP, ADP, and ATP) remain adsorbed to the alumina. The method permits the measurement of adenylylcyclase activity using [3H]ATP as the labeled substrate. The same technique can be used to measure the accumulation of cyclic AMP in intact cells after labeling the ATP pool with [3H]adenine. With slight modification, the technique can be used to measure the activity of cyclic-AMP phosphodiesterase using [3H]cyclic AMP as the substrate. The proposed technique provides rapid, highly reproducible assays using inexpensive, disposable columns.     

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%.     

Cyclic nucleotide phosphodiesterase. Partial purification and characterization of a high affinity enzyme activity from human lung tissue.

Part of the soluble cyclic nucleotide phosphodiesterase activity of crude human lung tissue can be attributed to a thermosensitive (37 degrees) enzyme with a high apparent affinity for both adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclic GMP). The enzyme can be partially purified by DEAE-Sephadex chromatography. In the presence of 0.1 mM EDTA or ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), it is eluted from the column immediately before a cyclic GMP-specific phosphodiesterase, but in the presence of 0.2 mM Ca2+, the elution follows that of the cyclic GMP-specific enzyme. The two forms of the nonspecific phosphodiesterase activity are referred to as DEAD-Sephadex Fractions Ia and Ic, respectively. Their apparent molecular weights, recorded at gel filtration, vary with different preparations from 230,000 to 150,000. Occasionally, corresponding recordings for main peaks of activity also cluster round the values 120,000, 105,000, and 78,000. The enzymatic properties of Fractions Ia and Ic closely resemble each other. The enzyme activity is blocked by EDTA, partially inhibited in the presence of 1,10-phenanthroline, but only slightly affected by EGTA. The inhibitory effect of EDTA can be overcome by Mg2+ and Mn2+ and that of 1,10-phenanthroline, in part, by Zn2+; this cation in itself is inhibitory at millimolar concentrations. With submicromolar substrate concentrations, the activity of either fraction obeys linear kinetics displaying an apparent Km of approximately 0.4 micron for both substrates. Reciprocal inhibition experiments suggest that hydrolysis of both cyclic AMP and cyclic GMP is performed by the same active site. Examination of the activity using extended substrate concentration ranges indicates nonlinear kinetics; Hill plots of such data also show nonlinear curvature. The activity is inhibited by micromolar concentrations of inosine 3':5'-monophosphate (cyclic IMP), 3-isobutyl-1-methylxanthine, papervine, and some antiallergic agents. Theophylline and disodium cromoglycate are less potent inhibitors. Inhibition of activity by Lubrol PX follows a biphasic dose response curve. The activity of Fraction Ia can be enhanced 2- to 3-fold by a Ca2+-dependent activator prepared from lung tissue, whose action is counteracted by chlorpromazine, and by lysophosphatidylcholine. It is initially enhanced but subsequently decreased at exposure to trypsin. Fraction Ic is less prone to activation by these agents. The results indicate that the present activity represents an enzyme form that differs from three previously described phosphodiesterases of human lung tissue. It is apparently related to, but also shows distinct differences from the Ca2+-dependent enzyme(s) of brain and heart tissue.