ADRENAL MEDULLARY CYCLIC NUCLEOTIDE PHOSPHODIESTERASE.—REGULATORY PROPERTIES OF THREE ENZYME ACTIVITIES

Abstract— Cyclic nucleotide phosphodiesterase from bovine adrenal medulla was fractionated into multiple activities by two different procedures, sucrose gradient centrifugation and gel filtration. Extracts of frozen and thawed adrenal medulla homogenates gave two phosphodiesterase activity peaks following density gradient centrifugation. The higher molecular weight activity hydrolyzed both cyclic AMP and cyclic GMP; ethylene glycol-bis(aminoethyl ether)- N,N' -tetraacetic acid (EGTA) inhibited only the hydrolysis of cyclic GMP. The lower molecular weight activity hydrolyzed only cyclic AMP and was not inhibited by EGTA. The two activities were not interconverted by recentrifugation.
Gel filtration of cyclic nucleotide phosphodiesterase activity extracted from frozen and thawed adrenal medulla on Ultrogel AcA 34 resolved the enzyme into three distinct peaks of enzyme activity with molecular weights of 350,000 (Peak I), 229,000 (Peak II) and 162,000 (Peak III). The enzyme from fresh tissue was resolved into peak I and II and only a small fraction of Peak III. Peak I hydrolyzed both cyclic nucleotides, while peak II was a cyclic GMP-specific enzyme and peak III was specific for cyclic AMP. The hydrolysis of cyclic AMP by the activity in Peak I was markedly stimulated by cyclic GMP; the hydrolysis of cyclic GMP by peak II was inhibited by EGTA and stimulated by calcium and CDR (calcium-dependent regulator protein). Peak III, which appears to be particulate, is not activated by either cyclic GMP or calcium and CDR.     

Cyclic nucleotide phosphodiesterase in silkworm. Separation and characterization of multiple forms of the enzyme.

The existence of two forms of cyclic AMP phosphodiesterase (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) was demonstrated in silkworm larvae by kinetic analysis and DEAE-cellulose column chromatography. The two forms of the enzyme (phosphodiesterase II and III) differ apparently in their characteristics from the previously reported cyclic nucleotide phosphodiesterase (phosphodiesterase I) of silkworm. The higher K-m form (phosphodiesterase II) has a molecular weight of approx. 50 000 and optimum pH of 7.8, and requires Mn-2-+ for maximum activity. The lower K-m form (phosphodiesterase III) has a molecular weight of approx. 97 000 and optimum pH of 7.2, and requires Mg-2-+ for maximum activity. Phosphodiesterase II and probably phosphodiesterase III are specific enzymes for the hydrolysis of cyclic AMP.     

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 activities of pig coronary arteries.

Most (85% or more) of the cyclic nucleotide phosphodiesterase (3' :5' -cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) activity of pig coronary arteries was found in the 40 000 times g supernatant fraction of homogenates of the intima plus media layer. Chromatography of the soluble fraction of this layer on DEAE-cellulose resolved two phosphodiesterase activities and a heat stable, non-dializable activator. Peak I activity had apparent Km values of 2-4 muM for cyclic GMP and 40-100 muM for cyclic AMP. Peak II activity was relatively specific for cyclic AMP and exhibited apparent negatively cooperative behavior. Peak I but not peak II activity could be stimulated 3-8-fold by the addition of the boiled activator fraction or a boiled crude supernatant fraction. Cyclic AMP hydrolysis by peak I or peak II was more rapid in the presence of Mn-2+ than Mg-2+, but the latter promoted hydrolysis of cyclic GMP by peak I more effectively than did Mn-2+ in the presence of activator. In the absence of added metals, ethylene bis(oxyethylenenitriol)tetra-acetic acid (EGTA) and EDTA both inhibited hydrolysis of cyclic AMP and cyclic GMP by phosphodiesterase activities in the supernatant fraction and in peak I, but EDTA produced more complete inhibition at lower concentrations than did EGTA. Imidazole (1 muM to 10 mM) had virtually no effect on the hydrolysis of cyclic AMP or cyclic GMP catalyzed by either of the two separated peaks or by total phosphodiesterase activities in crude supernatant or particulate fractions.     

3':5'-cyclic-nucleotide phosphodiesterase in the bovine pituitary gland

Cyclic-AMP phosphodiesterase activity in the homogenate of the anterior pituitary gland was 2-fold higher than that in the homogenate of the posterior pituitary, whereas cyclic-GMP phosphodiesterase activity was dominant in the posterior homogenate. There were two peaks of cyclic-AMP phosphodiesterase activity with different isoelectric points of 4.3 and 5.2. Fraction I had a molecular weight of 240 000 and a sedimentation coefficient of 6.2 S; fraction II had a molecular weight of 180 000 and a sedimentation coefficient of 3.1 S. Cyclic AMP hydrolytic activity in the supernatant of the posterior lobe corresponded to fraction I in the anterior lobe. Cyclic GMP hydrolytic activity in both the anterior and posterior lobes (activated by Ca2+/calmodulin) had an isoelectric point of 5.2, a molecular weight of 240 000 and a sedimentation coefficient of 6.2 S. Cyclic AMP and GMP hydrolytic activities in both the anterior and posterior lobes appeared in fraction I and did not separate when the preparations were mixed before electric focusing or sucrose density gradient procedures. Cyclic AMP hydrolytic activity in fraction II could be separated from cyclic GMP hydrolytic activity.     

3′:5′-Cyclic-nucleotide phosphodiesterase in the bovine pituitary gland

Cyclic-AMP phosphodiesterase activity in the homogenate of the anterior pituitary gland was 2-fold higher than that in the homogenate of the posterior pituitary, whereas cyclic-GMP phosphodiesterase activity was dominant in the posterior homogenate. There were two peaks of cyclic-AMP phosphodiesterase activity with different isoelectric points of 4.3 and 5.2. Fraction I had a molecular weight of 240 000 and a sedimentation coefficient of 6.2 S; fraction II had a molecular weight of 180 000 and a sedimentation coefficient of 3.1 S. Cyclic AMP hydrolytic activity in the supernatant of the posterior lobe corresponded to fraction I in the anterior lobe. Cyclic GMP hydrolytic activity in both the anterior and posterior lobes (activated by Ca²⁺ / calmodulin) had an isoelecteric point of 5.2, a molecular weight of 240 000 and a sedimentation coefficient of 6.2 S. Cyclic AMP and GMP hydrolytic activities in both the anterior and posterior lobes appeared in fraction I and did not separate when the preparations were mixed before electric focusing or sucrose density gradient procedures. Cyclic AMP hydrolytic activity in fraction II could be separated from cyclic GMP hydrolytic activity.     

Cyclic nucleotide phosphodiesterases of Hyalophora cecropia silkmoth fat body.

Two soluble forms of 3':5'-cyclic-nucleotide phosphodiesterase (o':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the larval fat body of the silkmoth Hyalophora cecropia. These differ in elution profile on Sephadex G-200, solubility in ammonium sulfate, metal ion requirements and kinetic properties. Phosphodiesterase I has Km values of 11 muM and 1.8 muM for cyclic AMP and cyclic GMP, respectively, has 5-fold greater maximal activity with cyclic AMP than with cyclic GMP, and is activated by Mg2+ and Co2+, and inhibited by EDTA. phosphodiesterase II has Km values of 625 muM and 125 muM for cyclic AMP and cyclic GMP, respectively, has similar maximal activity with both substrates, and is not activated by divalent metal ions or inhibited by EDTA. Cyclic nucleotides and methylxanthines competitively inhibit both enzymes. Phosphodiesterase is found in both soluble and particulate fractions of homogenates. Total activity is highest during the larval stage of the insect, drops markedly following pupation, and rises again during pharate adult development.     

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.     

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.     

Regulation by a β-adrenergic receptor of a Ca2+-independent adenosine 3′,5′-(cyclic)monophosphate phosphodiesterase in C6 glioma cells

The hormonal control of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity has been studied by using as a model the isoproterenol stimulation of cyclic AMP phosphodiesterase activity in C6 glioma cells. A 2-fold increase in cyclic AMP phosphodiesterase specific activity was observed in homogenates of isoproterenol-treated cells relative to control. This increase reached a maximum 3 h after addition of isoproterenol, was selective for cyclic AMP hydrolysis, was reproduced by incubation with 8-Br cyclic AMP but not with 8-Br cyclic GMP and was limited to the soluble enzyme activity. The presence of 0.1 mM EGTA did not alter the magnitude of the increase in phosphodiesterase activity. Moreover, the calmodulin content in the cell extracts was not changed after isoproterernol. DEASE-Sephacel chromatography of the 100 000×g supernatant resolved two peaks of phosphodiesterase activity. The first peak hydrolyzed both cyclic nucleotides and was activated by Ca²⁺ and purified calmodulin. The second peak was specific for cyclic AMP but it was Ca²⁺- and calmodulin-insensitive. Isoproterenol selectively increased the specific activity of the second peak. Kinetic analysis of the cyclic AMP hydrolysis by the induced enzyme reveled a non-linear Hofstee plot with apparent K_m values of 2–5 μM. Cyclic GMP was not hydrolyzed by this enzyme in the absence or presence of calmodulin and failed to affect the kinetics of the hydrolysis of cyclic AMP. Gel filtration chromatography of the induced DEASE-Sephacel peak resolved a single peak of enzyme activity with an apparent molecular weight of 54 000.     

Lack of altered cyclic nucleotide phosphodiesterase activity in the aorta and heart of the spontaneously hypertensive rat

DEAE-cellulose chromatography demonstrated that the levels of the individual cyclic nucleotide phosphodiesterase were unchanged in the aorta and heart of the spontaneously hypertensive rat as compared with the normotensive control rat. Three peaks of cyclic nucleotide phosphodiesterase activity were observed in both heart and aorta. Peak I enzyme hydrolyzed predominantly cyclic GMP while peak III enzyme hydrolyzed predominantly cyclic AMP. Peak II enzyme was less specific but hydrolyzed more cyclic GMP than cyclic AMP The levels of phosphodiesterase activator in aorta and the responsiveness of peaks I and II from aorta and heart to activator were unchanged in the hypertensive rat. Therefore the decrease in cyclic AMP levels observed by others in aorta and heart of the spontaneously hypertensive rat were probably not due to altered phosphodiesterase activity.     

Activation of mammalian cyclic AMP phosphodiesterases by trypsin.

BHK fibroblasts contain two forms of cyclic AMP phosphodiesterase 3':5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) as analyzed by linear sucrose gradient fractionation; a 3.6-S form (peak I) and a 6.7-S form (peak II). Peak I is specific for cyclic AMP as substrate and displays Michaelis-Menten kinetics with an apparent Km of 2--3 micrometer. Peak II hydrolyzes cyclic GMP and displays anomalous kinetics for cyclic AMP hydrolysis. The activity of isolated peak II for cyclic AMP is increased by storage at 4 degrees C, treatment with trypsin, or treatment with rat brain and BHK fibroblast activator proteins. The activity of isolated peak I is unaffected by these conditions. Linear sucrose gradient fractionation demonstrates that activation of peak II by trypsin leads to the formation of a 3.6-S cyclic AMP-specific enzyme form, possibly peak I. In contrast to BHK fibroblasts (and most other mammalian tissues), rat uterus contains only one form of cyclic nucleotide phosphodiesterase on linear sucrose gradients, a 7-S form capable of hydrolyzing both cyclic AMP and cyclic GMP. Treatment of rat uterine supernatant with trypsin leads to the appearance of a 4-S, cyclic AMP-specific form with properties similar to that of BHK peak I. These data suggest that the kinetically complex, higher molecular weight cyclic nucleotide phosphodiesterases may consist of more than one catalytically active site and that multiple forms of the enzyme arise through dissociative mechanisms, possibly as a means of in vivo regulation.     

Cyclic nucleotide phosphodiesterases from rat anterior pituitary. Characterization of multiple forms and regulation by protein activator and Ca+.

Phosphodiesterase activities for adenosine and guanosine 3':5'-monophosphates (cyclic AMP and cyclic GMP) were demonstrated in particulate and soluble fractions of rat anterior pituitary gland. Both fractions contained higher activity for cyclic GMP hydrolysis than that for cyclic AMP hydrolysis when these activities were assayed at subsaturating substrate concentrations. Addition of protein activator and CaCl2 to either whole homogenate, particulate or supernatant fraction stimulated both cyclic AMP and cyclic GMP phosphadiesterase activities. Almost 80% of cyclic AMP and 90% of cyclic GMP hydrolyzing activities were localized in soluble fraction. Particulate-bound cyclic nucleotide phosphodiesterase activity was completely solubilized with 1% Triton X-100. Detergent-dispersed particulate and soluble enzymes were compared with respect to Ca2+ and activator requirements and gel filtration profiles. Particulate, soluble and partially purified phosphodiesterase activities were also characterized in relation to divalent cation requirements, kinetic behavior and effects of Ca2+, activator and ethyleneglycol-bis-(2-aminoethyl)-N,N'-tetraacetic acid. Gel filtration of either sonicated whole homogenate or the 10500 X g supernatant fraction showed a single peak of activity, which hydrolyzed both cyclic AMP and cyclic GMP and was dependent upon Ca2+ and activator for maximum activity. Partially purified enzyme was inhibited by 1-methyl-3-isobutylxanthine and papaverine with the concentration of inhibitor giving 50% inhibition at 0.4 muM substrate being 20 muM and 24 muM for cyclic AMP and 7 muM and 10 muM for cyclic GMP, respectively. Theophylline, caffeine and theobromine were less effective. The rat anterior pituitary also contained a protein activator which stimulated both pituitary cyclic nucleotide phosphodiesterase(s) as well as activator-deficient brain cyclic GMP and cyclic AMP phosphodiesterases. Chromatography of the sonicated pituitary extract on DEAE-cellulose column chromatography resolved the phosphodiesterase into two fractions. Both enzyme fractions hydrolyzed cyclic AMP and cyclic GMP and had comparable apparent Km values for the two nucleotides. Hydrolysis of cyclic GMP and cyclic AMP by fraction II enzyme was stimulated 6--7-fold by both pituitary and brain activator in the presence of micromolar concentrations of Ca2+.     

Cyclic AMP-binding proteins and protamine kinases in porcine thyroid cytosol.

Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Ialpha and Ibeta) on DEAE-Sephadex A25. Fractions I, Ialpha, Ibeta comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the folling characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [3H]AMP showed similar mobilities for Ialpha, Ibeta or II (Rf 0.37) whereas fraction III displayed a much greater mobility (RF 0.73); Scatchard plots were linear for fractions Ialpha, II and III with an apparent Kd in the same range (2 to 5 nM) whereas fraction Ibeta generated a biphasic plot with Kd 0.4 nM and 20 nM; cyclic [3H] AMP added to fraction I, Ialpha or Ibeta generated a cyclic [3H] AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.     

Cyclic 3',5'-AMP phosphodiesterase of rabbit aorta.

Cyclic AMP and cyclic GMP phosphodiesterase activities (3' : 5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca2+ in the presence of the activator. However, the activity of the cyclic AMP phosphodiesterase was not significantly stimulated by Ca2+ up to 100 muM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60-70%) of the media enzyme was found in 105 000 times g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 times g supernatant the main kinetic parameters were specified as follows: 1) The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg2+ was achieved at 4mM of MgC12. 2) High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3) Activity-substrate concentration relationship revealed a high affinity (Km equals 1.0 muM) and low affinity (Km equals 45 muM) for cyclic AMP. The homogenate and 105 000 times g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent Km values for cyclic AMP hydrolysis were 1.2 muM and 36-40 muM and an enzyme extracted by sonication from 105 000 times g precipitate also exhibited non-linear kinetics (Km equals 5.1 muM and 70 muM). 4) Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I5 o at 0.62 muM, I5 o of the media at 0.62 muM and I5 o of the adventitia at 1.0 muM) than on the brain (I5 o at 8.5 muM) and serum (I5 o at 20 muM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.     

Regulation by insulin of cyclic nucleotide phosphodiesterase (PDE) from rat luteal cells

The effect of insulin on cyclic nucleotide phosphodiesterase (PDE) in rat luteal cells was studied. Cells were obtained from PMSG/hCG primed rats and further incubated or not with insulin. The hormone produced an increase of enzyme activity after a 10 min incubation of intact cells. Maximal stimulation was achieved at 0.2 nM of insulin. Two peaks of cyclic nucleotide phosphodiesterase activity were resolved after chromatography of cell cytosolic extracts on DEAE-cellulose. These peaks (I and II) were active with cAMP as substrate but only peak I was active with cGMP. The enzyme activity of both peaks was increased in cells treated with insulin. Phosphodiesterase activity in the two peaks show two kinetic components for cAMP hydrolysis, one of high affinity (Km 2-4 microM) and the other of low affinity (47-56 microM). Treatment of the cells with insulin produced a 2 to 8 fold increase of the Vmax of these peaks. In addition after stimulation with insulin, the activation of peak I phosphodiesterase by calmodulin was less effective.     

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

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

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.     

Function of calmodulin in postsynaptic densities. I. Presence of a calmodulin-activatable cyclic nucleotide phosphodiesterase activity

The postsynaptic density (PSD) fraction from canine cerebra cortex was found to contain an endogenous cyclic nucleotide-phosphodiesterase activity that was independent on Mn2+ and/or Mg2+ but not on Ca2+. Maximal activity was obtained at 1 micrometer Mn2+. This cyclic nucleotide phosphodiesterase activity was not decreased upon removal of the calmodulin from the PSD fraction, nor was it increased by the addition of calmodulin to a postsynaptic density fraction deficient in calmodulin. The enzymatic activity could be extracted by sonication, with the soluble enzyme having properties similar to those found in the native structure. Two peaks of cyclic nucleotide phosphodiesterase activities could be obtained after S-300 Sephacryl column chromatography of this soluble fraction: fraction I (excluded peak) and fraction II (215,000 mol wt). The fraction I activity preferred cyclic AMP over cyclic GMP and was not activated by calmodulin. The fraction II activity has an approximately fourfold lower Km for cyclic GMP over cyclic AMP. This fraction II activity was activatable by calmodulin, which increased the Vmax and decreased the Km in the case of both cyclic nucleotides. We conclude that two activities are present in the PSD, one activatable, and one not activatable, by calmodulin.     

Multiple cyclic nucleotide phosphodiesterase activities from rat tissues and occurrence of a calcium-plus-magnesium-ion-dependent phosphodiesterase and its protein activator.

1. Supernatant fluids from rat cerebral cortex, cerebellum, kidney, heart and liver contained more phosphodiesterase activity hydrolysing cyclic GMP than that hydrolysing cyclic AMP when assayed with sub-saturating concentrations of substrate. 2. These activities were resolved into several fractions by Sephadex G-200 gel filtration; no two tissues had similar activity profiles. 3. With every tissue examined, a fraction (fraction II) with a molecular weight of about 150,000 was obtained which hydrolysed cyclic GMP preferentially at sub-saturating substrate concentrations in the presence of micromolar concentration of Ca2+, millimolar concentration of Mg2+ and a protein activator. 4. The activity of fraction II accounted for about 60 percent in liver, more than 80 percent in heart and cerebellum, and almost 100 percent in cerebral cortex of the total activity for cyclic GMP hydrolysis, calculated from the activity profiles. 5. Km values of fraction II samples from kidney, heart and liver for cyclic GMP were 1.3, 1.7 and 5 muM respectively. 6. 3-Isobutyl-1-methylxanthine inhibited hydrolysis of cyclic GMP by fraction II with an I50 value of 3muM for heart and liver and 50 muM for cerebrum. 7. The activator protein, with an estimated molecular weight of about 30,000 was isolated from all the tissues listed in 1.8. The concentrations of activator protein and of the isolated enzyme, fraction II, did not correspond exactly.