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

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

Properties of cyclic nucleotide phosphodiesterase of the rabbit myometrium in various functional states

Chromatography on DEAE-cellulose of a soluble sulfate-precipitated fraction of cyclic nucleotide phosphodiesterase from rabbit myometrium revealed two 3':5'-GMP and 3':5'-AMP-hydrolase activities. 3':5'-GMP phosphodiesterase (fraction I) was eluted with 0.15-0.23 M NaCl, while 3':5'-AMP phosphodiesterase (fraction II) with 0.2-0.35 M NaCl. 3':5'-GMP phosphodiesterase hydrolyzed 3':5'-GMP with Km = 14 microM and V = 5.25 nmol . min . mg of protein, while 3':5'-AMP phosphodiesterase hydrolyzed both cyclic nucleotides with Km for 3':5'-GMP equal to 12 microM and V = 1.33 nmol . min . mg of protein; the Km value for 3':5'-AMP was 3.6 and 30.5 microM, respectively; the corresponding values of V were 0.28 and 0.97 nmol . min . mg of protein. In late pregnancy, the level of the 3':5'-AMP hydrolase activity of rabbit myometrium was significantly elevated in parallel with an increase in V, predominantly for the enzyme with a low affinity for 3':5'-AMP. The 3':5'-GMP hydrolase activity and V were largely decreased for both phosphodiesterase fractions; the Km value for fraction I was also diminished. During labour, the rate of 3':5'-AMP hydrolysis by myometrium phosphodiesterase was decreased down to the level typical of functional rest. The rate of 3':5'-GMP hydrolysis during the same period by fraction I remained at a low level, i. e., as in pregnancy, while that of fraction II was increased up to the level typical of functional rest.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Cyclic nucleotide phosphodiesterase from wheat sprouts. Purification, properties, effect of systemic fungicides

Cyclic nucleotide phosphodiesterase from wheat sprouts was isolated and partially purified. The molecular weight of the enzyme is about 83 000. The enzyme activity sharply rises as the inhibiting factors present in the homogenate are separated. The pH optimum of the enzymatic reaction is 4,8. Divalent cations (Mg2+, Mn2+, Cu2+) within the concentration range of 1--5 mM and complexons (EDTA, EGTA) at the concentration of 1 mM do not affect the PDE activity. The temperature optimum for the reaction is 60 degrees. The enzyme hydrolyzes 3' : 5'-AMP, 3' : 5'-GMP and 2':3'-AMP. The Km value for cAMP is 4 . 10(-3) M. The enzyme activity is inhibited by chemical agents possessing the fungicide activity, the strongest effect being exerted by anylate.     

rac-Glycerol 1:2-cyclic phosphate 2-phosphodiesterase, a new soluble phosphodiesterase of mammalian tissues.

1. A soluble phosphodiesterase is present in mammalian tissues which rapidly hydrolyses enantiomorphs of rac-glycerol 1:2-cyclic phosphate, producing rac-glycerol 1-phosphate. 2. The enzyme has been purified up to 1700-fold by a combination of acetone precipitation and chromatography on DEAE-Sephadex A-50, Sephadex G-150 and hydroxyapatite. 3. The Km with glycerol cyclic phosphate as substrate is 7.2 mM, and the pH optimum broad (6.9--7.5). The molecular weight (by gel filtration) of the enzyme is approx. 35500. 4. The phosphodiesterase has no requirement for Ca2+ or Mg2+, but is stimulated by reducing agents (cysteine, dithiothreitol) and Fe2+. 5. The purified phosphodiesterase preparation also hydrolysed 3':5'-cyclic AMP, producing 5'-AMP exclusively, and 2':3'-cyclic AMP, forming 3'-AMP and 2'-AMP in the ratio 7:3. Bis-(p-nitrophenyl) phosphate was slowly hydrolysed, but other phosphodiesters tested were not attacked. 6. The phosphodiesterase is inhibited by theophylline and o-phenanthroline. It is inhibited by Pi and by a variety of phosphomonoesters, of which certain aromatic primary phosphates are particularly effective.     

Isolation and properties of 2 forms of cyclic nucleotide phosphodiesterase from the rat brain under normal conditions and after irradiation

It is established that the functional activity of two phosphodiesterase forms--phosphodiesterase I (Ca2+-calmodulin-sensitive) and phosphodiesterase II (Ca2+-calmodulin-insensitive), isolated from grey matter of the irradiated rat brain varies essentially in comparison with that of the normal rats. In the early period of acute radiation injury both phosphodiesterase I sensitivity to calmodulin and phosphodiesterase II special activity under hydrolysis of 3', 5'-GMP decrease but phosphodiesterase I special activity under hydrolysis of 3', 5'-GMP increases. The investigation of temperature dependence of phosphodiesterase I and phosphodiesterase II activations revealed changes in character of curves, the temperature optimum under irradiation being unchanged and inflections appearing on the Arrhenius curves.     

Repressible extracellular phosphodiesterases showing cyclic 2'',3''- and cyclic 3'',5''-nucleotide phosphodiesterase activities in Neurospora crassa.

Two molecular species of repressible extracellular phosphodiesterases showing cyclic 2',3'- and cyclic 3',5'-nucleotide phosphodiesterase activities were detected in mycelial culture media of wild-type Neurospora crassa and purified. The two molecular species were found to be monomeric and polymeric forms of an enzyme constituted of identical subunits having molecular weights of 50,000. This enzyme had the same electrophoretic mobility as repressible acid phosphatase. The enzyme designated repressible cyclic phosphodiesterase showed pH optima of 3.2 to 4.0 with a cyclic 3',5'-AMP substrate and 5.0 to 5.6 with a cyclic 2',3'-AMP substrate. Repressible cyclic phosphodiesterase was activated by MnCl2 and CoCl2 with cyclic 2',3'-AMP as substrate and was slightly activated by MnCl2 with cyclic 3',5'-AMP. The enzyme hydrolyzed cyclic 3',5'- and cyclic 2',3'-nucleotides, in addition to bis-rho-nitrophenyl phosphate, but not certain 5' -and 3'-nucleotides. 3'-GMP and 3'-CMP were hydrolyzed less efficiently. Mutant strains A1 (nuc-1) and B1 (nuc-2), which cannot utilize RNA or DNA as a sole source of phosphorus, were unable to produce repressible cyclic phosphodiesterase. The wild type (74A) and a heterocaryon between strains A1 and B1 produced the enzyme and showed growth on orthophosphate-free media containing cyclic 2',3'-AMP or cyclic 3',5'-AMP, whereas both mutants showed little or no growth on these media.     

The isoenzyme selectivity of AH 21-132 as an inhibitor of cyclic nucleotide phosphodiesterase activity.

The smooth muscle relaxant, AH 21-132, was tested for its inhibitory effect on the cyclic nucleotide phosphodiesterase (PDE) activities fractionated from guinea-pig cardiac ventricle and bovine trachealis muscle. Both tissues yielded significant PDE-I and PDE-II activities. The cardiac ventricle also contained a significant amount of PDE-III whilst the trachealis contained PDE-IV. AH 21-132 inhibited PDE-III and PDE-IV selectively (Ki values 0.30-0.55 microM) compared with PDE-I and PDE-II (Ki values 20-140 microM).     

Regulation of urocaninase activity in the liver: role of 3',5'-AMP]

A dependence of rat liver urocaninase activity on the agents affecting the adenylate cyclase system was studied in vitro and in vivo. Urocaninase is considerably activated after the injection of glucagone, NaF, theophylline and 3',5'-AMP. Under conditions optimal for the protein kinase activity of phosphorylase the urocaninase of liver extracts was activated 7-fold on the average. The nezyme retains its activity after gel-filtration through Sephadex G-25 and is capable of inactivation in the presence of Mg2+ and of reactivation after addition of ATP and 3',5'-AMP. These data suggest a possibility of regulation of mammalian liver urocaninase activity by 3',5'-AMP-dependent phosphorylation of the enzyme. Derivatives of hypoxanthine (theophylline and caffeine) in concentration 10(-4) M activate urocaninase in liver extracts 2--3 and 1.5-fold respectively. The activation is probably not due to the 3',5'-AMP phosphodiesterase inhibition, since another phosphodiesterase inhibitor--papaverine--has no activating effect on urocaninase.     

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 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.     

Partial purification and characterization of adenosine- and guanosine-3',5'-monophosphate phosphodiesterases from human lung tissue.

Crude extracts of human lung tissue were examined for cyclic adenosine- and guanosine-3',5'-monophosphate (cAMP and cGMP) phosphodiesterase activities. Nonlinear reciprocal plots were observed for each substrate. DEAE-Sephadex chromatography of the extracts revealed four main fractions of activity, which were further purified by Sephadex gel filtration. The phosphodiesterase activity of the resulting individual fractions was partially characterized with respect to substrate specificity, kinetic parameters, apparent molecular weight (gel filtration), thermal stability at 30 and 37 degrees C, effect of the cyclic nucleotide not utilized as substrate, and the possible influence of Ca2+-dependent protein activator. The results indicate that the tissue contains phosphodiesterases with strict specificity and a high apparent affinity for each of the two cyclic nucleotides (the Km values determined were approximately 0.3-0.4 muM). The high affinity cAMP phosphodiesterase activity was enriched in two of the purified fractions; both activities probably represent fragments of the native high affinity cAMP specific enzyme. A third purified phosphodiesterase showed mixed substrate specificity. The Km value recorded for hydrolysis of either substrate with this enzyme was approximately 25 muM. A fourth, irregularly occurring, phosphodiesterase activity also showed mixed substrate specificity. The Km value registered for hydrolysis of either substrate with this fraction was approximately 0.4 muM. There was no evidence for a Ca2+-dependent specific activation by a boiled lung tissue supernatant of any of the purified enzymes.     

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%.     

Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts.

Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.     

Purification and characterization of a cyclic nucleotide-regulated 5'-nucleotidase from potatoe.

A procedure is presented for the rapid purification of a 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) from potato tubers, involving ammonium sulphate fractionation and chromatography on phosphocellulose, DEAE-cellulose and Sephadex G-75. Application of this procedure results in a 6000-fold purification of the 5'-nucleotidase and the final preparations are virtually homogeneous, yielding only one protein band on electrophorsis in polyacrylamide gels in non-dissociating or dissociating conditions. The 5'-nucleotidase has a molecular weight of 50 000 from gel filtration experiments. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified 5'-nucleotidase reveals one major band of molecular weight 25 000. The 5'-nucleotidase is competitively inhibited by cyclic nucleotides, having micromolar Ki values for cyclic AMP and cyclic GMP at pH 5.0 and pH 8.0. The enzyme has a pH optimum of 5.0 with 5'-GMP as substrate. While 5'-AMP and 3'-AMP are hydrolyzed at comparable rates at pH 5.0, at pH 8.0 the rate of hydrolysis of 3'-AMP is only 4% of that with 5'-AMP. ADP, ATP and 2'-AMP are very poor substrates for the enzyme. The nucleotidase has micromolar Km values for nucleoside 5'-monophosphates other than 5'-NMP. A wide variety of divalent cations activate the 5'-nucleotidase.     

The identification and characterization of two cyclic nucleotide phosphodiesterases from bovine adrenal medulla

Two soluble cyclic nucleotide phosphodiesterase activities, designated Peak I (Mr = 216,000) and Peak II (Mr = 230,000), have been isolated from bovine adrenal medulla by DEAE-cellulose chromatography. Peak I has Ca2+-independent, cGMP-specific phosphodiesterase activity and Peak II has cGMP-stimulated cyclic nucleotide phosphodiesterase activity. Peak I hydrolyzes cGMP with hyperbolic kinetics and demonstrates a Km of 23 microM. Peak II hydrolyzes cGMP with hyperbolic kinetics but hydrolyzes cAMP with slightly sigmoidal kinetics and demonstrates Km values of 54 +/- 0.7 microM cGMP and 38 +/- 6 microM cAMP. Cyclic AMP and cGMP are competitive inhibitors of each other's hydrolysis, suggesting that these nucleotides may be hydrolyzed at the same catalytic site. Micromolar concentrations of cGMP cause a 5-fold stimulation of the hydrolysis of subsaturating concentrations of cAMP by the Peak II phosphodiesterase. Half-maximal activation occurs at 0.5 microM cGMP and the result of activation is a decrease in the apparent Km for cAMP. Stimulation of the hydrolysis of subsaturating concentrations of cGMP by cAMP was also detected; however, cAMP is a less potent activator of the enzyme than cGMP. Cyclic AMP causes a 1.5-fold stimulation of cGMP hydrolysis and half-maximal activation occurs at 2.5 microM cAMP.     

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.     

A phosphate-binding histidine of binuclear metallophosphodiesterase enzymes is a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity

Binuclear metallophosphoesterases are an enzyme superfamily defined by a shared fold and a conserved active site. Although many family members have been characterized biochemically or structurally, the physiological substrates are rarely known, and the features that determine monoesterase versus diesterase activity are obscure. In the case of the dual phosphomonoesterase/diesterase enzyme CthPnkp, a phosphate-binding histidine was implicated as a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity. Here we tested this model by comparing the catalytic repertoires of Mycobacterium tuberculosis Rv0805, which has this histidine in its active site (His(98)), and Escherichia coli YfcE, which has a cysteine at the equivalent position (Cys(74)). We find that Rv0805 has a previously unappreciated 2',3'-cyclic nucleotide phosphodiesterase function. Indeed, Rv0805 was 150-fold more active in hydrolyzing 2',3'-cAMP than 3',5'-cAMP. Changing His(98) to alanine or asparagine suppressed the 2',3'-cAMP phosphodiesterase activity of Rv0805 without adversely affecting hydrolysis of bis-p-nitrophenyl phosphate. Further evidence for a defining role of the histidine derives from our ability to convert the inactive YfcE protein to a vigorous and specific 2',3'-cNMP phosphodiesterase by introducing histidine in lieu of Cys(74). YfcE-C74H cleaved the P-O2' bond of 2',3'-cAMP to yield 3'-AMP as the sole product. Rv0805, on the other hand, hydrolyzed either P-O2' or P-O3' to yield a mixture of 3'-AMP and 2'-AMP products, with a bias toward 3'-AMP. These reaction outcomes contrast with that of CthPnkp, which cleaves the P-O3' bond of 2',3'-cAMP to generate 2'-AMP exclusively. It appears that enzymic features other than the phosphate-binding histidine can influence the orientation of the cyclic nucleotide and thereby dictate the choice of the leaving group.     

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.     

Crystal and Molecular Structures of Monomethyl Esters of PDE-I and PDE-II,New Inhibitors of Cyclic Adenosine-3′,S′-monophosphate Phosphodiesterase

The structures of PDE-I and PDE-II, inhibitors of cyclic adenosine-3′,5′-monophosphate phosphodiesterase, were established as 3-carbamoyl-l,2-dihydro-4-hydroxy-5-methoxy-3-H-pyrrolo[3,2-e]indole-7-carboxylic acid and its 3-acetyl homologue, by X-ray diffraction study of monomethyl derivatives.