Cyclic nucleotide phosphodiesterases and thyroid hormones.

Evidence is presented that modulation of the maximum velocity of a particulate low K-m cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase by thyroid hormones is one mechanism for the regulation of the responsiveness of rat epididymal adipocytes to lipolytic agents such as epinephrine and glucagon. Fat cells of propylthiouracil-induced hypothyroid rats are unresponsive to lipolytic agents and the V-max of particulate low K-m cyclic AMP phosphodiesterase of these cells is elevated above normal. In vivo treatment of hypothyroid rats with triiodothyronine restores to control values both the lipolytic response of the fat cells to epinephrine and the V-max of the particulate bound low K-m cyclic AMP phosphodiesterase. No similar correlation is found with the soluble high K-m cyclic AMP phosphodiesterase. The phosphodiesterases of fat cells from normal and hypothyroid rats respond identically in vitro to propylthiouracil, triiodothyronine, methylisobutylxanthine, or theophylline, although the particulate low K-m cyclic AMP phosphodiesterase is inhibited to a greater extent than soluble cyclic guanosine 3':5'-monophosphate phosphodiesterase activity. Protein kinase of fat cells from hypothyroid rats can be stimulated by cyclic AMP to the same total activity as observed in fat cells of normal rats. However, less of the protein kinase in fat cells from hypothyroid rats was in the cyclic AMP-independent form. This shift in the equilibrium of protein kinase forms is consistent with an increased activity of low K-m cyclic AMP phosphodiesterase and probably results from a lowering of the lipolytically significant pool 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 in Neurospora crassa.

Cyclic nucleotide phosphodiesterase activities in soluble Neurospora crassa mycelial extracts were resolved into two peaks, phosphodiesterase I and II, by chromatography on DEAE-cellulose columns. Phosphodiesterase I hydrolysed cyclic AMP and cyclic GMP equally well. Phosphodiesterase II was active on cyclic GMP but scarcely active on cyclic AMP. Phosphodiesterase I was resolved by gel filtration and sucrose-density-gradient centrifugation into three peaks having molecular weights of about 57 000, 125 000 and 225 000. This suggests that this enzyme activity has at least three aggregation forms, tentatively defined as monomeric, dimeric and tetrameric. Similarly, phosphodiesterase II was resolved into two forms, having molecular weights of about 170 000 and 320 000. Evidence on the interconversion between phosphodiesterase I forms was obtained.     

Characterisation of cyclic adenosine 3':5'-monophosphate phospodiesterase from Walker carcinoma sensitive and resistant to bifunctional alkylating agents.

Walker carcinoma cell lines sensitive or resistant to bifunctional alkylating agents have been found to contain multiple forms of cyclic AMP phosphodiesterase (3':5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). These activities have been resolved using Sepharose 6B gel filtration and their apparent molecular weights have been estimated. The enzyme appears to occur in four active forms of apparent mol. wts of greater than 1 000 000, 430 000, 350 000 and 225 000, when assayed at low substrate concentrations. Evidence has been obtained which suggests that all four forms of the enzyme are composed of subunits of mol. wt of approximately 15 000 and are interconvertible. While the ionic strength of the buffer affected the predominance of the different forms, the presence of cyclic AMP at 10(-6) M had no effect on aggregation or dissociation of the enzyme. An activity shift from high molecular weight forms of the enzyme to low molecular weight forms has been found in the resistant tumour at low substrate concentration. No change in elution profile between sensitive and resistant tumours was observed for the low affinity form of the enzyme. The pH optima of the enzymes with both high and low affinity for the substrate was found to be pH 8.0 in the sensitive line. In the resistant tumour the pH optima of the high affinity form is shifted to pH 8.4 while the low affinity form remains at pH 8.0. The high affinity forms of the phosphodiesterase in the sensitive and resistant tumour also differed in their inhibition by theophylline. In both cases inhibition was of the competitive type with Ki values for the sensitive and resistant lines being 2.35 and 0.32 mM, respectively. There was no significant difference in the inhibition of the low affinity form between the sensitive and resistant tumour.     

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.     

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.     

Purification of an alkaline ribonuclease from soluble fraction of beef brain.

A ribonuclease (ribonucleate 3-pyrimidine-oligonucleotidohydrolase, EC 3.1.4.22) was purified 8300-fold from soluble fraction of beef brain and its properties were investigated. The enzyme is an endonuclease capable of hydrolyzing tRNA, rRNA, poly(C), but shows no activity towards poly(U), poly(A), and poly(G). The preparation is free of deoxyribonuclease, non-specific phosphodiesterase and phosphomonoesterase activity. The enzyme has a pH optimum of 7.6, is not heat stable, has a molecular weight of 25 000, and has a K-m of 134 mu rRNA and K-m of 1600 mug poly(C) per ml.     

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.     

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.     

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.     

Adenosine cyclic 3',5'-monophosphate-dependent protein kinase from human platelets.

A single cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been isolated from human platelets by using DEAE-cellulose ion-exchange chromatography and Sephadex G-150 gel filtration. The molecular weight of the protein kinase was estimated to be 86 490. In the presence of cyclic AMP, the protein kinase could be dissociated into a catalytic subunit of molecular weight 50 000, and either one regulatory subunit of molecular weight 110 000 or two regulatory subunits of molecular weights 110 000 and 38 100, depending on the pH used. Recombination of either of the regulatory subunits with the catalytic subunit restored cyclic AMP-dependency in the catalytic subunit. The apparent Km for ATP in the presence of 10 muM Mg2+ was 4 muM (plus cyclic AMP) and 4.3 muM (minus cyclic AMP). The concentration of cyclic AMP needed for half-maximal stimulation of the protein kinase was 0.172 muM and apparent dissociation constants of 3.7 nM (absence of MgATP) and 0.18 muM (presence of MgATP) were exhibited by the "protein kinase-cyclic AMP complex". The enzyme required Mg2+ for maximum activity and showed a pH optimum of 6.2 with histone as substrate. In addition to four major endogenous platelet protein acceptors of apparent molecular weights 45 000, 28000, 18 500, and 11 100, the platelet protein kinase also phosphorylated the exogenous acceptor proteins thrombin, collagen and histone, all capable of inducing platelet aggregation. Prothrombin, a nonaggregating agent, was not phosphorylated.     

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 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.     

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.     

Human thyroid cyclic nucleotide phosphodiesterase. Its characterization and the effect of several hormones on the activity.

Cyclic AMP and cyclic GMP phosphodiesterase activities (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were investigated in the human thyroid gland from patients with hyperthyroidism. Low substrate concentration (0.4 muM) was used. About 60% of the cyclic-AMP and 80% of the cyclic-GMP hydrolytic activities in the homogenate were obtained in the soluble fraction (105 000 X g supernatant). The thyroid gland contains two forms of cyclic-AMP phosphodiesterase, one with a Km of 1.3-10(-5) M and the second with a Km of 2-10(-6) M. Cyclic-AMP and cyclic-GMP phosphodiesterase were purified by gel filtration on a Sepharose-6B column. Cyclic-AMP phosphodiesterase activities were found in a broad area corresponding to molecular weights ranging from approx. 200 000 to 250 000 and cyclic-GMP phosphodiesterase activity was found in a single area corresponding to a molecular weight of 260 000. Cyclis-AMP phosphodiesterase activities were stimulated by the protein activator which was found in human thyroid and this stimulation was dependent on Ca2+. Stimulation of cyclic-AMP phosphodiesterase by the activator was not significant even in the presence of enough Ca2+. The effect of D,L-triiodothyronine, D,L-thyroxine, L-diiodotyrosine, L-monoiodotyrosine, L-thyronine, L-diiodothyronine, thyrotropin, hydrocortisone, adrenocorticotropin, cyclic-AMP and cyclic-GMP on the phosphodiesterase activities was studied. Cyclic-AMP, cyclic-GMP, D,L-triiosothyronine, D,L-thyroxine, adrenocorticotropin and hydrocortisone where found to inhibit the phophodiesterase. Triiodothyronine and thyroxine inhibited cyclic-AMP phosphodiesterase more effectively than cyclic-GMP phosphodiesterase. Thyroxine was a more potent inhibitor than triiodothyronine. The concentration of cyclic AMP producing a 50% inhibition of cyclic-GMP phosphodiesterase activity was 5-10(-5) M, while the concentration of cyclic GMP producing a 50% inhibition of cyclic-AMP phosphodiesterase was 3-10(-3) M. Both cyclic-AMP and cyclic-GMP phosphodiesterase activities in the homogenate of hyperthyroidism, thyroid carcinoma and adenoma were higher than in normal thyroid tissue, when assayed with a low concentration of the substrate (0.4 muM). When a higher concentration (1 mM) of cyclic nucleotides was used as the substrate, cyclic-AMP hydrolytic activity in adenoma tissue was similar to that of normal tissue, while the other activities were higher than normal.     

Cyclic nucleotide phosphodiesterases in the cricket, Acheta domesticus.

Exceptionally high levels of guanosine 3'-5'-cyclic monophosphate (cyclic GMP) in the accessory reproductive gland of the male house cricket, Acheta domesticus, led to an investigation of cyclic nucleotide phosphodiesterase (EC 3.1.4.--) as a possible regulatory enzyme. Cricket cyclic nucleotide phosphodiesterase activity with cyclic GMP or cyclic AMP as substrate had a pH optimum around 9.0, required Mg2+ or Mn2+ for maximal activity, and was inhibited by EDTA and methylxanthines. Cyclic GMP phosphodiesterase occurred mainly in the soluble fraction of homogenates of accessory glands or whole crickets, but cyclic AMP phosphodiesterase in the accessory gland was primarily particulate. Kinetic analysis indicated three forms of cyclic GMP phosphodiesterase, with Km values at 2.9 muM, 71 muM and 1.5 mM. Chromatography of whole cricket or accessory gland extracts on DEAE cellulose gave an initial peak having comparable activity with either cyclic GMP or cyclic AMP, and a second peak specific for cyclic AMP. There were no appreciable changes in the specific activity or kinetic properties of accessory gland cyclic GMP phosphodiesterase during a developmental period over which cyclic GMP levels rise more than 500-fold. Thus, the accumulation of cyclic GMP in the accessory gland is probably not associated with concomitant developmental modulation of phosphodiesterase activity.     

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.     

Effect of Ricinus communis toxin on cyclic adenosine 3':5'-monophosphate metabolism in Yoshida ascites sarcoma cells.

Prostaglandin E1 (2.5 mug/ml) enhanced the level of cyclic adenosine 3':5'-monophosphate (cyclic AMP) three to four times in Yoshida ascites sarcoma (YS) cells cultured in vitro. When Ricinus communis toxin (RC-toxin) was added 30 min after the addition of prostaglandin E1, the enhanced level of cyclic AMP in the YS cells decreased rapidly. Of RC-toxin, 0.2 mug/ml was enough to produce the maximum effect. By addition of 5 mM lactose with RC-toxin, approximately 60% of the RC-toxin effect on the levels of cyclic AMP was abolished. This indicates that the specific binding of RC-toxin on the surface membrane is largely responsible for the observed decrease of the cyclic AMP level. The toxin treatment did not induce either leakage of cyclic AMP from the cell or change in the activity of cyclic AMP phosphodiesterase. However, the treatment of YS cells with RC-toxin caused a decrease of adenylate cyclase activity when the activity was measured at a substrate concentration of 0.15 mM ATP. In contrast, there was little difference with the control when the activity was assayed at a higher ATP concentration, 0.24 mM. It was found that the K-m of adenylate cyclase for ATP was changed by RC-toxin from 0.1 to 0.25 mM, and that the Mg2+ activation of the enzyme observable in untreated cells disappeared. These results suggested that the decrease in the level of cyclic AMP in YS cells induced by RC-toxin can be explained in terms of the change in K-m of the adenylate cyclase activity.     

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.     

Investigations on adenosine 3',5'-monophosphate phosphodiesterase in ram semen and initial characterization of a sperm-specific isoenzyme.

Phosphodiesterase is shown to occur in ram semen, and its activity to be higher in spermatozoa than in seminal plasma. Using similar substrate levels, the rate at which adenosine 3',5'-monophosphate (cyclic AMP) is metabolized by phosphodiesterase in spermatozoa is about 100 times higher than that of cyclic AMP synthesis by adenylate cyclase. In spermatozoa, phosphodiesterase is present partly in a soluble form, and partly bound; both forms can be extracted by sonication. The soluble enzyme (pH optimum 8-0, Km = 1-5 muM, mol. wt 165,000) occurs as a single isoenzyme, as shown by polyacrylamide gel electrophoresis and anion-exchange chromatography; this isoenzyme appears to be specific for spermatozoa and its formation in the testis coincides with the appearance of spermatozoa. The bound sperm enzyme has been solubilized with Trion X-100; it is a single isoenzyme (pH optimum 8-0, mol. wt 165,000) which is electrophoretically different from the soluble form, but similar to the phosphodiesterase found in other tissues. Seminal plasma phosphodiesterase (pH optimum 8-8, mol. wt 165,000) is present in the form of three isoenzymes; all three are different from the two forms of sperm phosphodiesterase, but are similar to the isoenzymes found in certain male accessory organs.