Purification and characterization of wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase

The 2',3'-cyclic nucleotide 3'-phosphodiesterase which hydrolyzes nucleoside 2',3'-cyclic phosphates (N greater than p) to nucleoside 2'-phosphates has been purified 16,000-fold to near homogeneity from wheat germ. The purified enzyme is a single polypeptide with a molecular weight of 23,000-24,000. It has a pH optimum of 7.0. The apparent Km values for A greater than p, G greater than p, C greater than p, and U greater than p are 13.1, 9.2, 25.2, and 25.3 mM, respectively. Vmax values for A greater than p, G greater than p, C greater than p, and U greater than p are 2090, 280, 2140, and 600 mumol/min/mg of purified protein, respectively. Wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase does not hydrolyze 2',3'-cyclic esters in cyclic phosphate-terminated oligoribonucleotides or in nucleoside 5'-phosphate, 2',3'-cyclic phosphate (pN greater than p). This is in contrast to the 3'-phosphodiesterase activity associated with a wheat germ RNA ligase which hydrolyzes cyclic phosphate-terminated oligonucleotides and pN greater than p substrates much more efficiently than nucleoside 2',3'-cyclic phosphates. The enzyme characterized in this work appears to be the only known 2',3'-cyclic nucleotide 3'-phosphodiesterase specific for 2',3'-cyclic mononucleotides.     

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.     

Enzymology and genetic regulation of a cyclic nucleotide-binding phosphodiesterase-phosphomonoesterase from Aspergillus nidulans.

A cyclic nucleotide-binding phosphohydrolase that possesses both a phosphomonoesterase and a phosphodiesterase catalytic function has been partially purified from Aspergillus nidulans. The enzyme hydrolyzes both p-nitrophenylphosphate and bis-(p-nitrophenyl)-phosphate. o'-Nucleoside monophosphates are the best physiological phosphomonesterase substrates but 5'- and 2'-nucleoside monophosphates are also hydrolyzed. The enzyme catalyzes the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and 2',3'- and 3'5'-cyclic nucleotides, but not of ribonucleic acid, deoxyribonucleic acid, or nicotinamide adenine dinucleotide. The enzyme has acid pH optima and is not activated by divalent cations. Nucleosides and nucleotides inhibit the enzyme. Cyclic nucleotides are competitive inhibitors of the phosphodiesterase-phosphomonoesterase. The enzyme can occur extracellularly. The phosphodiesterase-phosphomonoesterase is present at high levels in nitrogen-starved mycelium, and it is strongly repressed during growth in media containing ammonium or glutamine and weakly repressed during growth in glutamate-containing medium. Experiments with various area mutants show that this regulatory gene is involved in the control of the enzyme. No evidence for regulation of the enzyme by carbon or phosphorus starvation has been found.     

Properties of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium.

The properties of three phosphatases from Salmonella typhimurium have been examined. A cyclic 2',3'-nucleotide phosphodiesterase (EC 3.1.4.d) hydrolyzes cyclic 2',3'-purine and -pyrimidine nucleotides, as well as 3'-mononucleotides, and has a pH optimum of about 7.5. It requires divalent cations for activity and has a molecular weight of 67,000. Acid hexose phosphatase (EC 3.1.2.2) possesses activity towards hexose phosphates as well as other sugar phosphates. The enzyme is apparently a dimer of 37,000-dalton subunits. Nonspecific acid phosphatase (EC 3.1.3.2) hydrolyzes a variety of phosphate esters, including nucleotides and sugar phosphates. The enzyme also hydrolyzes the phosphoric anhydride bonds of pyrophosphate and nucleotides. Michaelis constants of the nonspecific acid phosphatase for several of its substrates are in the 1 to 2 mM range. Nonspecific acid phosphatase is a dimer of 27,000-dalton subunits.     

2',3'-seco pyrimidine nucleosides and nucleotides, including structural analogues of 3':5'-cyclic CMP and UMP, and their behaviour in several enzyme systems

Cyclization of 2',3'-seco-5'- CMP and UMP with dicyclohexylcarbodiimide leads to 2',3'-seco-3':5'- cCMP and cUMP, formal structural analogues of 3':5'- cCMP and cUMP. POCl3 phosphorylation of 2',3'-secocytidine gave the same product in 50% yield, plus three additional seco nucleotides, one of which was independently obtained by enzymatic phosphorylation with the wheat shoot phosphotransferase system. The behaviour of these nucleotides has been examined in several enzyme systems. In particular, the seco 3':5'- cyclic phosphates are resistant to beef heart cyclic nucleotide phosphodiesterase, but are slowly hydrolyzed to the monophosphates by higher plant cyclic nucleotide phosphodiesterase.     

A simple direct assay of 3',5'-cyclic nucleotide phosphodiesterase activity based on the use of polyacrylamide-bononate affinity gel chromatography.

A rapid, simple, and direct assay for 3',5'-cyclic nucleotide phospho-diesterase activity is based on the effective separation of cyclic AMP, cyclic GMP or cyclic CMP from their corresponding 5'-nucleotides and nucleosides by chromatography on a polyacrylamide-boronate gel. The affinity of the boronate residue for cis-diols results in the retention of 5'nucleotides and nucleosides while 3',5'-cyclic nucleotides are not retained. The coelution of all 5'-nucleotides and nucleosides allows for the accurate assessment of phosphodiesterase activity in preparations contaminated by other purine metabolizing enzymes such as 5'-nucleotidases and nucleotide and nucleoside deaminases. Phosphodiesterase activity assayed by this means yields linear reaction kinetics with respect to time and amount of enzyme protein. Low blank values obtained allow for detection of as little as 2-3% conversion of substrate to product.     

Determination of cyclic nucleotide phosphodiesterase activity by high-performance liquid chromatography

A new assay for cyclic nucleotide phosphodiesterase activity by high-performance liquid chromatography with on-line radiochemical detection has been developed. The method is based on the measurement of 3H-labeled nucleoside monophosphates formed from cyclic nucleotides by the action of 3',5'-cyclic-nucleotide phosphodiesterase (PDE). The reaction products are determined from the incubation mixture after removal of the protein by injection of an aliquot into the liquid chromatograph. The detection limit with counting efficiency of 30% is 20 fmol of 3H-labeled product, which makes the method suitable for detection of low PDE activities.     

2'',3''-CYCLIC NADP AS A SUBSTRATE FOR 2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASE

Abstract– 2',3'-Cyclic NADP has been prepared by cyclization of NADP at pH 6 in the presence of l-ethyl-(3-dimethylaminopropyl)-carbodiimide. The NADP derivative is readily hydrolyzed to NADP by the enzyme in brain and nerve that hydrolyzes 2',3'-cyclic nucleotides to 2'-phospho esters. The K _m for this substrate is the same as that for 2',3'-cyclic AMP (0.22 m m ) at pH 6 and 25°C. The two substrates are hydrolyzed by the phosphohydrolase at similar maximum velocities. The nicotinamide moiety in cyclic NADP thus has little effect on the enzyme-substrate interaction. This synthetic substrate can be used in a rapid (2 min) and sensitive (10 ng of 31-fold purified enzyme) spectrophotometric coupled enzyme assay for 2',3'-cyclic nucleotide 3'-phosphohydrolase; in this assay the hydrolysis proceeds in the presence of glucose-6-phosphate dehydrogenase and its substrate and the NADPH formed is measured by the increase in absorbance at 340 nm. The assay is applicable to tissue extracts as well as to purified preparations of the enzyme. There is no interference from nucleases of the pancreatic RNase A type.     

Kinetic studies on degradation of nucleotides catalyzed by phosphodiesterase-phosphomonoesterase from Fusarium moniliforme

Degradation of the 2'-phosphates, 3'-phosphates, 5'-phosphates, 2':3'-cyclic phosphates, 3':5'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) of adenosine, guanosine, cytidine, and uridine catalyzed by Fusarium phosphodiesterase-phosphomonoesterase was followed by means of high performance liquid chromatography. All the nucleotides were susceptible to the enzyme to a greater or lesser degree, and the kinetic constants, Km and kcat, were determined at pH 5.3 and 37 degrees C. These constants were affected by both the nucleoside moiety and the position of the phosphate. Judged from kcat/Km, the 3'-phosphates, 2':3'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) were good substrates, whereas the 2'-phosphates, 5'-phosphates, and 3':5'-cyclic phosphates were poor substrates except for adenosine 2'-phosphate, adenosine 5'-phosphate, and cytidine 5'-phosphate, which were hydrolyzed relatively easily. Among the phosphodiesters, the 2':3'-cyclic phosphates of adenosine, guanosine, and cytidine; and the 3':5'-cyclic phosphates of adenosine and cytidine were degraded into nucleoside and inorganic phosphate without release of intermediary phosphomonoester into the medium. Other phosphodiesters were degraded stepwise releasing definite intermediates.     

Temperature sensitivity of cyclic-adenosine-3':5'-monophosphate-binding proteins, activity of protein kinases and the regulation of cell growth.

A clone of neuroblastoma cells has been selected for its ability to survive and multiply at 40 degrees C. This temperature-resistant clone, like clones of neuroblastoma cells selected for resistance to dibutyryladenosine 3':5'-monophosphate (Bt2-Ado-3':5'-P) showed an increased tumorogenicity in animals and an increased saturation density at 37 degrees C. The Ado-3':5'-P-binding proteins and Ado-3':5'-P-dependent protein kinases from the temperature-resistant and non-resistant cells have been partially purified by chromatography on a DEAE-cellulose column. The Ado-3':5'-P-binding proteins from temperature-resistant cells were more sensitive to temperature than the binding proteins from non-resistant cells. After incubation of binding proteins from resistant cells at 37 degrees C, the specific activity of Ado-3':5'-P-binding to proteins was decreased about 50% and the apparent association constant (Ka) for Ado-3':5-p-binding was decreased from 7.4 X 10(7)M-1 to 4.4 x 10(7)M-1. There was no such decrease with binding proteins from non-resistant cells. A decrease in the activity of binding proteins from the temperature-resistant cells, but not of those from non-resistant cells, was also found when the proteins were stored at 2 degrees C. Treatment with 2-mercaptoethanol made binding proteins from the resistant cells less temperature-sensitive. In the absence of added Ado-3:5-P the protein kinase activity from the temperature-resistant cells was about 50% of the activity from non-resistant cells. Kinase activity was increased by addition of Ado-3:5-P and there was a greater increase with kinases from resistant cells. The maximum protein kinase activity was found in the presence of 10muM Ado-3':5'-P for the temperature-resistant cells and 0.1 muM Ado-3':5'-P for the non-resistant cells. The results indicate that the temperature sensitivity of Ado-3':5'-P-binding proteins, and the activity of protein kinase from cells selected for resistance to high temperature, are similar to those of cells selected for resistance to Bt2-Ado-3':5'-P. It is suggested that the temperature sensitivity of Ado-3':5'-P-binding proteins and the activity of Ado-3':5'-P-dependent protein kinases are involved in the regulation of malignancy and of cell growth at different temperatures.     

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.     

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.     

A comparative kinetic study of bovine calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes utilizing cAMP, cGMP and their 2'-O-anthraniloyl-,2'-O-(N-methylanthraniloyl)-derivatives as substrates

Calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase (EC 3.1.4.17) Mr 63,000 and Mr 60,000 from the brain as well as Mr approximately 59,000 species from the heart, have been compared with respect to their steady-state kinetic parameters for the hydrolysis of cAMP, cGMP and their 2'-O-anthraniloyl- and 2'-O-(N-methylanthraniloyl)-derivatives. Kinetic studies with the native substrates indicate high Mr brain enzyme to be cGMP specific whereas low Mr brain and heart enzymes to be nonspecific. In addition, the isozymes studied here appear to be kinetically distinct from those previously isolated form bovine brain tissues. Substitution at 2'-O-position of the cyclic nucleotides gave rise to Vmax values ranging 1-11% of those observed with the native substrates, with minimal effect on Km. The isozymes with exception of heart isoform gave higher Km and Vmax with the anthraniloyl derivatives. This effect is thought to be related to the formation of an intramolecular hydrogen bond which leads to decreased electrostatic interactions between the active-site side chains and the pseudo-substrates.     

The number and role of histidine residues in the active site of guanyloribonuclease Sa

The number and role of histidine residues in the active site of extracellular guanyloribonuclease Sa produced by Streptomyces aureofaciens (RNAase Sa) were studied via chemical modification by ethoxyformic anhydride by means of circular dichroism measurements. It was shown that only one of two histidines of RNAase Sa is situated in the active site of the enzyme. Ethoxyformylation of RNAase Sa in the presence of Guo-3'-P, Guo-5'-P and dGuo-5-P, all of them being competitive inhibitors of the enzyme, supported the assumption that an essential histidine residue is bound to the phosphate group in the position 3' of the ribose ring. The circular dichroism measurements of native and modified RNAase Sa and of its complex with Guo-3'-P showed that the modification of the essential histidine residue resulted in alteration of binding of RNAase Sa to Guo-3'-P; histidine thus may play a key role in the formation of such a complex.     

Isolation and characterization of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum

A new enzyme that hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate has been purified by a factor of 250 from the acellular slime mold Physarum polycephalum. Activity was assayed radioisotopically with [3H]Ap4A. Isolation of the enzyme was facilitated by dye-ligand chromatography. The enzyme symmetrically hydrolyzes Ap4A to ADP and exhibits biphasic kinetics for the substrate with values for the apparent Km of 2.6 micro M and 37 micro M. The two values of Vmax differ by a factor of 10. Mg2+, Ca2+, and other divalent cations inhibit the activity with 40-80% inhibition occurring at 0.5 mM. Mg2+, at 0.5 mM, decreases both values of Vmax by 50%, decreases the low Km value by about 30%, and increases the high Km value by about 100%. (Ethylenedinitrilo)tetraacetic acid (EDTA) and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), at 10 mM, inhibit the activity by 50%. ADP, ATP, Ap4, and Gp4 are equipotent inhibitors with 50% inhibition occurring at 30 micro M. AMP is a relatively weak inhibitor. The molecular weight of the enzyme is 26000 on the basis of elution of activity from a calibrated Sephadex G-75 column.     

Enzymes hydrolyzing ApppA and/or AppppA in higher plants. Purification and some properties of diadenosine triphosphatase, diadenosine tetraphosphatase, and phosphodiesterase from yellow lupin (Lupinus luteus) seeds

Three distinct enzymes hydrolyzing either ApppA or AppppA, or both, were separated and purified from yellow lupin seed extracts. Two of the enzymes were purified to homogeneity. These enzymes differ greatly in their catalytic and physical properties. One hydrolase, with a native molecular weight of 41,000, exhibits broad pH (from 5-8) optimum for activity, requires Mg2+ for activity, is inhibited by zinc ions (I0.5 = 25 microM) and hydrolyses ApppA (V = 1), ApppC (V = 0.38), ApppG (V = 0.2), and ribose(5')pppA (V = 0.2). The enzyme exhibits much lower activity with AppppA (V = 0.1), and ApppppA, AppppppA, ppppA, and ATP are hydrolyzed 25- to 100-fold slower then ApppA. ADP was always one of the products of the reactions catalyzed by the enzyme. AppA, NAD, NADP, FAD, cAMP, and p-nitrophenyl-thymidine 5'-phosphate were not hydrolyzed by the enzyme. The enzyme is diadenosine 5',5"'-P1, P3-triphosphatase. The second hydrolase, composed of one polypeptide chain of a molecular weight 18,000-18,500, exhibits optimal activity in the pH range from 7.5-9, requires Mg2+ for activity, is inhibited by calcium ions (I0.5 for calcium depends on the concentration of Mg2+ and is 35-180 microM in the presence of 0.5-10 mM Mg2+, respectively), and hydrolyzes AppppA (V = 1, Km = 1 microM), ApppppA (V = 0.42, Km = 1.8 microM), AppppppA (V = 0.34), AppppU (V = 0.73), AppppC (V = 0.67), AppppG (V = 0.27), and ppppA. ATP was always one of the products of the reactions catalyzed by the enzyme. Dinucleoside di- and triphosphates, ATP, cAMP, and p-nitrophenylthymidine 5'-phosphate were not hydrolyzed by the enzyme. This enzyme is diadenosine 5',5"'-P1,P4-tetraphosphatase (EC 3.6.1.17). The third hydrolase, composed of one polypeptide chain of a molecular weight of 56,000, exhibits maximal activity at pH 9-10.5, does not require Mg2+ ions for activity, is inhibited neither by divalent cations (Mg2+, Ca2+, Zn2+, Co2+, Mn2+, or Ni2+) nor by EDTA, and uses as substrates all compounds which are substrates for the diadenosine 5',5"'-P1,P3-triphosphatase and diadenosine 5',5"'-P1,P4-tetraphosphatase. In addition, the enzyme hydrolyzes p-nitrophenyl-thymidine 5'-phosphate, p-nitrophenylthymidine 3'-phosphate, bis-p-nitrophenylphosphate, ADP, AppA, NAD, NADP, and FAD, but not cAMP. With the exception of p-nitrophenylphosphate derivatives all other substrates of the enzyme yield AMP as one of the products of hydrolysis. This enzyme has a specificity similar to that of phosphodiesterases (EC 3.1.4.1) from other sources. With the lupin phosphodiesterase, ApppA (V = 1, Km = 2.2 microM) and AppppA (V = 1, Km = 2.0 microM) are better substrates than NAD (V = 0.8, Km = 9.6 microM), AppA (V = 0.4), ApppppA (V = 0.6), and AppppppA (V = 0.34).     

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%.     

Alkaline ribonuclease from rye germ cytosol.

1. Alkaline ribonuclease (pH optimum 7.6) was isolated from rye (Secale cereale L) germ cytosol and partially purified; the preparation was devoid of other nucleolytic activities. 2. The enzyme is a typical endonuclease hydrolysing all phosphodiester bonds in RNA, yielding ultimately purine and pyrimidine nucleoside 2',3'-cyclic phosphates and the corresponding 3'-phosphates. Upon extensive digestion of synthetic polyribonucleotides, pyrimidine, but not purine, nucleoside 3'-phosphates are formed. The enzyme does not hydrolyse synthetic purine cyclic nucleotides. 3. The enzyme does not depolymerize double-stranded complexes of poly(A) and poly(U). 4. Susceptibility to photooxidation and inhibition by 2-hydroxy-5-nitrobenzyl bromide and N-bromosuccinimide implies the involvement of tryptophan residue in the active centre of the enzyme.     

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.     

Cyclic nucleotide content of tobacco BY-2 cells

The cyclic nucleotide content of cultured tobacco bright yellow-2 (BY-2) cells was determined, after freeze-killing, perchlorate extraction and sequential chromatography, by radioimmunoassay. The identities of the putative cyclic nucleotides, adenosine 3',5'-cyclic monophosphate (cyclic AMP), guanosine 3',5'-cyclic monophosphate (cyclic GMP) and cytidine 3',5'-cyclic monophosphate (cyclic CMP) were unambiguously confirmed by tandem mass spectrometry. The potential of BY-2 cell cultures as a model system for future investigations of cyclic nucleotide function in higher plants is discussed.