Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber.

1. Three phosphodiesterases that are capable of hydrolysing 3':5'-cyclic nucleotides were purified from potato tubers. 2. The phosphodiesterases were fractionated by (NH4)2SO4 precipitation and CM-cellulose chromatography. The phosphodiesterases were resolved from each other and further purified by gel filtration in high- and low-ionic-strength conditions. 3. All three enzymes lacked significant nucleotidase activity. 4. Enzymes I and II had mol. wts. 240,000 and 80,000 respectively, determined by gel filtration, whereas enzyme III showed anomalous behaviour on gel filtration, behaving as a high- or low-molecular-weight protein in high- or low-ionic-strength buffers respectively. 5. All enzymes hydrolysed 2':3'-cyclic nucleotides as well as 3':5'-cyclic nucleotides. The enzymes also had nucleotide pyrophosphatase activity, hydrolysing NAD+ and UDP-glucose to various extents. Enzymes I and II hydrolyse cyclic nucleotides at a greater rate than NAD+, whereas enzyme III hydrolyses NAD+ at a much greater rate than cyclic nucleotides. All three enzymes hydrolysed the artificial substrate bis-(p-nitro-phenyl) phosphate. 6. The enzymes do not require the addition of bivalent cations for activity. 7. Both enzymes I and II have optimum activity at pH6 with 3':5'-cyclic AMP and bis-(p-nitrophenyl) phosphate as substrates. The products of 3':5'-cyclic AMP hydrolysis were 3'-AMP and 5'-AMP, the ratio of the two products being different for each enzyme and varying with pH. 8. Theophylline inhibits enzymes I and II slightly, but other methyl xanthines have little effect. Enzymes I and II were competitively inhibited by many nucleotides containing phosphomonoester and phosphodiester bonds, as well as by Pi. 9. The possible significance of these phosphodiesterases in cyclic nucleotide metabolism in higher plants is discussed.     

Studies of the 2':3'-cyclic nucleotide phosphodiesterase of Haemophilus influenzae

The 2':3'-cyclic nucleotide phosphodiesterase:3'-nucleotidase of Haemophilus influenzae was purified from a periplasmic preparation by affinity chromatographic techniques. The enzyme-catalysed hydrolysis of 2':3'-cyclic AMP to adenosine without accumulation of the intermediate substrate 3'-AMP was demonstrated by high performance liquid chromatography. Competitive inhibition of the enzyme by a variety of nucleosides and mononucleotides indicated the presence of either purine or pyrimidine bases to be essential for selective interactions with the enzyme, and confirmed the need for a 3'-position phosphate for the functioning of mononucleotides as substrates for the enzyme. The enzyme had a molecular weight of 79 000, was stable at low temperatures and was thermally denatured at temperatures above 50 degrees C.     

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.     

Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2':3'-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970-976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2':3'-cyclic NMP to 3'-NMP and the 3':5'-cyclic isomers to a mixture of 3'-NMP and 5'-NMP. Specific activities of the enzyme are 2-fold higher for 2':3'-cyclic NMP than for 3':5'-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared -Pi culture medium as a source of 3'-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.     

A stereochemical investigation of the hydrolysis of cyclic AMP and the (Sp)-and (Rp)-diastereoisomers of adenosine cyclic 3'':5''-phosphorothioate by bovine heart and baker''s-yeast cyclic AMP phosphodiesterases.

Bovine heart cyclic AMP phosphodiesterase, which has a requirement for Mg2+, hydrolyses cyclic AMP with inversion of configuration at the phosphorus atom, but only the (Sp)-diastereoisomer of adenosine cyclic 3':5'-phosphorothioate is hydrolysed by this enzyme. By contrast, the low-affinity yeast cyclic AMP phosphodiesterase, which contains tightly bound Zn2+, hydrolyses both the (Sp)- and the (Rp)-diastereoisomers of adenosine cyclic 3':5'-phosphorothioate, the (Rp)-diastereoisomer being the preferred substrate under V max. conditions. Both of the diastereoisomers of adenosine cyclic 3':5'-phosphorothioate, as well as cyclic AMP, are hydrolysed with inversion of configuration at the phosphorus atom by the yeast enzyme. It is proposed that, with both enzymes, the bivalent metal ion co-ordinates with the phosphate residue of the substrate, and that hydrolysis is catalysed by a direct "in-line' mechanism.     

Isolation of a 5.3-S calmodulin-deficient 3'':5''-cyclic nucleotide phosphodiesterase from cardiac muscle.

1. In the presence of Ca2+, a 5.3-S 3':5'-cyclic nucleotide phosphodiesterase (EC 3.1.4.17) from bovine ventricle was isolated and purified by (NH4)2SO4 precipitation and DEAE-cellulose and Affi-Gel Blue chromatography. The enzyme activity was enriched 800-fold by these procedures. 2. Sucrose-density gradient centrifugation, gel filtration and non-denaturing polyacrylamide-gel electrophoresis resolved a single enzyme species with an Mr of 89 000. 3. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the purified enzyme demonstrated a prominent protein band at Mr 59000 and a minor band of Mr 28000. Calmodulin was not detected. 4. The hydrolysis of micromolar concentrations of 3':5'-cyclic guanosine monophosphate (cyclic GMP) but not 3':5'-cyclic adenosine monophosphate (cyclic AMP) was stimulated by calmodulin. 5. Anomalous biphasic kinetics plots were observed for both the catalysis of cyclic AMP and cyclic GMP hydrolysis. Kinetic plots became linear in the presence of calmodulin. 6. After several months of storage at -20 degrees C, the 5.3-S enzyme was transformed into a 6.2-S cyclic GMP-specific enzyme and a 4.4-S non-specific form.     

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.     

A comparison of d-inositol 1:2-cyclic phosphate 2-phosphohydrolase with other phosphodiesterases of kidney

1. The ability to hydrolyse various phosphodiesterase substrates was examined in subcellular fractions of rat kidney and in serial slices of the kidneys of mouse, rat, guinea pig and ox cut from the cortex perimeter inwards. 2. d-Inositol 1:2-cyclic phosphate 2-phosphohydrolase could be clearly distinguished from phosphodiesterases which hydrolyse 2':3'- and 3':5'-cyclic AMP and p-nitrophenyl thymidine 5'-phosphate (phosphodiesterase I). The hydrolysis of sn-glycero-3-phosphorylcholine showed a distribution identical with that of particle-bound d-inositol 1:2-cyclic phosphate 2-phosphodiesterase, but there was a 30-fold difference in the ratio of enzyme activities between the rat and guinea pig. 3. In rat and mouse kidney, d-inositol 1:2-cyclic phosphate 2-phosphohydrolase is virtually all membrane bound and in the outer cortex, whereas in guinea-pig kidney the enzyme is almost entirely soluble and located throughout the kidney tissue. Some properties of the soluble enzyme are described. 4. Distribution and histochemical studies indicated that in the rat and mouse, phosphodiesterase I is associated with the brush borders of the straight portion (pars recta) of the proximal tubule, whereas inositol 1:2-cyclic phosphate 2-phosphohydrolase and probably glycerylphosphorylcholine diesterase are associated with the brush borders of the convoluted part of the tubule (pars convoluta).     

Cyclic 3',5'-AMP phosphodiesterase in Physarum polycephalum. I. Chemotaxis toward inhibitors and cyclic nucleotides.

The effect of several inhibitors of the enzyme cyclic 3',5'-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3',5'-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3',5'-AMP at concentrations as high as 1 . 10(-2) M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3',5'-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3',5'-AMP in the directional movement in P. polycephalum is discussed.     

The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives

The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue.     

The influence on platelet aggregation of drugs that affect the accumulation of adenosine 3′:5′-cyclic monophosphate in platelets

1. The involvement of intracellular 3':5'-cyclic AMP in the inhibition of platelet aggregation by prostaglandin E(1), isoprenaline and adenosine has been examined by a radiochemical technique. Platelet-rich plasma was incubated with radioactive adenine to incorporate (14)C radioactivity into platelet nucleotides. Pairs of identically treated samples were taken, one for the photometric measurement of platelet aggregation induced by ADP, the other for estimation of the radioactivity of 3':5'-cyclic AMP. 2. Theophylline, papaverine, dipyridamole and 2,6-bis-(diethanolamino)-4-piperidinopyrimido[5,4d]pyrimidine (compound RA233) were found to inhibit 3':5'-cyclic AMP phosphodiesterase from platelets. At concentrations of 3':5'-cyclic AMP greater than 50mum the most active inhibitor was dipyridamole; at 3':5'-cyclic AMP concentrations less than 19mum, papaverine and compound RA233 were more active than dipyridamole. 3. In the presence of compound RA233 (50mum), the effectiveness of prostaglandin E(1) as an inhibitor of platelet aggregation was increased tenfold. Compound RA233 also increased the stimulation by prostaglandin E(1) of the incorporation of radioactivity into 3':5'-cyclic AMP. 4. Compound RA233 (50mum) increased the effectiveness of both adenosine and 2-chloroadenosine as inhibitors of aggregation by 70-100-fold, and in the presence of compound RA233 both adenosine and 2-chloroadenosine stimulated the incorporation of radioactivity into 3':5'-cyclic AMP; the extent of the stimulation was proportional to the logarithm of the nucleoside concentration. 5. Compound RA233 (100-500mum) inhibited platelet aggregation by itself and caused small increases in the radioactivity of 3':5'-cyclic AMP. Partial positive correlations were found between the radioactivity of 3':5'-cyclic AMP in platelets measured at the time of addition of the aggregating agent (ADP) and the extent to which the aggregation was inhibited. 6. The results are interpreted as indicating that adenosine, 2-chloroadenosine, isoprenaline, prostaglandin E(1) and drugs that inhibit platelet 3':5'-cyclic AMP phosphodiesterase all inhibit aggregation by a common mechanism involving intracellular 3':5'-cyclic AMP.     

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.     

Effects of adenosine 5'-monophosphate and adenosine 3',5'-cyclic monophosphate on l cells.

The adenine nucleotides, 5'-AMP and 3',5'-cyclic AMP block L cells in the S-phase of the cell cycle. The intracellular level of cyclic AMP is reduced after incubation of cells with 5'-AMP, and rates of uridine transport are increased after incubation with either 5'-AMP or cyclic AMP. On the contrary, cyclic AMP levels are increased and uridine transport decreased in cells treated with an inhibitor of the cyclic AMP phosphodiesterase. This inhibitor partially reverses the growth-inhibitory effect of cyclic AMP, indicating that a breakdown product is the effective inhibitor of growth. The inhibition of cell growth induced by the adenine nucleotides is prevented by uridine, suggesting that the block in S is due to a lack of availability of pyrimidines.     

Characterization of the soluble cyclic nucleotide phosphodiesterases in Xenopus laevis oocytes. Evidence for a calmodulin-dependent enzyme

Cyclic nucleotide phosphodiesterase activities (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the 40,000 X g supernatant fraction of homogenates of Xenopus laevis oocytes. In the supernatant, the ratio of the specific activity of cyclic AMP phosphodiesterase to that of cyclic GMP phosphodiesterase was 1.1 at the 1 micro substrate level. Two phosphodiesterase forms were isolated by centrifugation on sucrose gradient: a 3-4 S form hydrolyzing specificity cyclic AMP and a 6-7 S form hydrolyzing both cyclic nucleotides (cyclic AMP and cyclic GMP). The activity of the 6-7 S phosphodiesterase was characterized by its activation by 0.1 micro M calmodulin purified from beef pancreas in the presence of 50 micro M CA2+. The calmodulin dependence of this form was completely abolished in the presence of 1 mM ethyleneglycobis(beta-aminoethyl ether)-N-N,N',N'-tetraacetic acid (EGTA). Trifluoperazine at 0.1 mM inhibited both the freshly prepared crude enzyme and the partially purified 6-7 S form. On the other hand, no effect of cyclic GMP at 3 micro M was observed on cyclic AMP hydrolysis in the case of the supernatant or that of the partially purified phosphodiesterases. These data show the presence of a calmodulin-dependent phosphodiesterase in the soluble fraction of X. laevis oocytes.     

Effect of cyclic 3':5'-AMP derivatives prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture.

The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N6, O2'-dibutyryl cyclic 3':5'-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process, malignant trophoblast cultures were incubated with 3':5'-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmunoassayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretion of hCG, the N6--and O2'-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3':5'-GMP (cGMP), dbcGMP, 5'-AMP, adenosine, L-epinephrine and prostaglandins E1, E2, F1a and F2a were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast.     

Hydrolysis of phosphonate esters catalyzed by 5'-nucleotide phosphodiesterase.

4-Nitrophenyl and 2-napthyl monoesters of phenylphosphonic acid have been synthesized, and an enzyme catalyzing their hydrolysis was resolved from alkaline phosphatase of a commerical calf intestinal alkaline phosphatase preparation by extensive ion-exchange chromatography, chromatography on L-phenylalanyl-Sepharose with a decreasing gradient of (NH4) 2SO4, and gel filtration. Detergent-solubilized enzyme from fresh bovine intestine was purified after (NH4)2SO4 fractionation by the same technique. The purified enzyme is homogeneous by polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. It has a molecular weight of 108,000, contains approximately 21% carbohydrate, and has an amino acid composition considerably different from that reported from alkaline phosphatase from the same tissue. The homogeneous intestinal enzyme, an efficient catalyst of phosphonate ester hydoolysis but not of phosphate monoester hydrolysis, was identified as a 5'-nucleotide phosphodiesterase by its ability to hydrolyze 4-nitrophenyl esters of 5'-TMP but not of 3'-TMP. Also consistent with this identification was the ability of the enzyme to hydrolyze 5'-ATP to 5'-AMP and PPi, NAD+ to 5'-AMP and NMN, TpT to 5'-TMP and thymidine, pApApApA to 5'-AMP, and only the single-stranded portion of tRNA from the 3'-OH end. Snake venom 5'-nucleotide phosphodiesterase also hydrolyzes phosphonate esters, but 3'-nucleotide phosphodiesterase of spleen and cyclic 3',5'-AMP phosphodiesterase do not. Thus, types of phosphodiesterases can be conveniently distinguished by their ability to hydrolyze phosphonate esters. As substrates for 5'-nucleotide phosphodiesterases, phosphonate esters are preferable to the more conventional esters of nucleotides and bis(4-nitrophenyl) phosphate because of their superior stability and ease of synthesis. Furthermore, the rate of hydrolysis of phosphonate esters under saturating conditions is greater than that of the conventional substrates. At substrate concentrations of 1 mM the rates of hydrolysis of phosphonate esters and of nucleotide esters are comparable and both superior to that of bis(4-nitrophenyl) phosphate.     

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%.     

2',3'-cyclic nucleotide 2'-phosphodiesterase from Fusarium culmorum

We describe the properties of a 2',3'-cyclic nucleotide 2'-phosphodiesterase (EC 3.1.4.16), found in Fusarium culmorum, which hydrolyzes nucleoside 2',3'-cyclic monophosphates to nucleoside 3'-phosphates. In contrast with a similar enzyme found in bacteria, the Fusarium enzyme does not exhibit nucleotidase activity and does not show a requirement for metal ions, but is inhibited by micromolar concentrations of Cu++ and Zn++, and is very stable to heat. This cyclic phosphodiesterase hydrolyzes the four major nucleoside 2',3'-cyclic monophosphates and has greater affinity for purine (Kms for Ado-2',3'-P = 0.3 mM and for Guo-2',3'-P = 0.1 mM) than for pyrimidine nucleotides (Kms for Cyd-2',3'-P = 0.6 mM and for Urd-2',3'-P = 2 mM). The respective Vmax for Urd-2',3'-P; Cyd-2',3'-P; Ado-2',3'-P; and Guo-2',3' are 100:45:16:5. The efficacy of the phosphodiesterase to hydrolyze the four major 2',3' cyclic nucleotides (based on the relative values of Vmax/Km) is not significantly different. The Fusarium enzyme differs from a previously described 2',3' cyclic phosphodiesterase from Neurospora, in that it is inactive on 3',5'-nucleoside monophosphates and nucleoside 2' or 3' phosphates.     

Rat intestinal phosphodiesterase II. Properties of the highly purified enzyme and its inactivation by iodoacetic acid.

A highly purifed preparation of rat intestinal phosphodiesterase II (oligonucleate 3'-nucleotidohydrolase, EC 3.1.4.18) has been studied using a synthetic substrate, thymidine 3'(2,4-dinitrophenyl) phosphate. The enzyme was most active between pH 6.1 and pH 6.7 and was inhibited by Cu2+ and Zn2+ but unaffected by EDTA, Mg2+, Co2+, and Ni2+. The reaction rate decreased at high levels of enzyme because of competitive inhibition by deoxythymidine 3'-phosphate, a reaction product, which showed a Ki of 2-10(-5) M. The molecular weight of the enzyme by gel-filtration was 150 000-170 000. In electrofocusing experiments multiple peaks of activity were found at pH 3.4, 4.2-4.5and 7.2. Polyacrylamide gel electrophoresis of freshly purified phosphodiesterase II showed up to 10 protein bands in the gels. If the preparations were stored at 4 degrees C for some time only one or two bands appeared. Investigation of the reaction of rat intestinal phosphodiesterase II with a number of possible phosphodiesterase substrates indicated that the enzyme required a nucleoside 3'-phosphoryl residue for the initiation of hydrolysis. Thus compounds such as NAD, ATP, bis-(p-nitrophenyl)phosphate, thymidine 5'-(p-nitrophenyl)phosphate, glycerylphosphorylcholine, guanylyl-(2' leads to 5')-adenosine and 3',5'-cyclic AMP which contain phosphodiester bonds, nevertheless were not substrates for the enzyme. The enzyme was inhibited reverisbly by p-chloromercuribenzoate and p-chloromercuriphenylsulfonate and inactivated irreversibly by iodoacetic acid. Activity of the phosphodiesterase II was reduced to 50% by incubation with 2.0-10(-3)--5.0-10(-3) M iodoacetate for 20--30 min at 24 degrees C at pH 5.0--6.1. Iodoacetamide had no effect. The degree of inactivation by iodoacetate was reduced by the presence of a substrate for the enzyme or, more effectively by deoxythymidine 3'-phosphate, a competitive inhibitor. It is concluded that iodoacetic acid alkylates an essential residue at the active centre of the enzyme.     

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.