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.     

3',5'-Cyclic nucleotide phosphodiesterase activity of the sulphatase A of ox liver

The sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver hydrolyses adenosine 3',5'-monophosphate (cyclic AMP) to adenosine 5'-phosphate at an optimum pH of approx. 4.3, close that for the hydrolysis of cerebroside sulphate, a physiological substrate for sulphatase A. The Km is 11.6 mM for cyclic AMP. On polyacrylamide gel electrophoresis sulphatase A migrates as a single protein band which coincides with both the arylsulphatase and phosphodiesterase activities, suggesting that these are due to a single protein. Cyclic AMP competitively inhibits the arylsulphatase activity of sulphatase A, showing that both activities are associated with a single active site on the enzyme. sulphatase A also hydrolyses guanosine 3',5'-monophosphate, but not uridine 3',5'-monophosphate nor adenosine 2',3'-monophosphate.     

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.     

Purification of rat heart cyclic nucleotide phosphodiesterase on column of immobilized phenylbutenolide inhibitor

Cyclic nucleotide phosphodiesterase was purified over 200-fold in a single step from the rat heart cytosolic fraction, using affinity chromatography on phenylbutenolide inhibitor immobilized to AH Sepharose. After elimination of the contaminating proteins by washing with the loading buffer and then with 0.4 M KCl buffer, without any loss in enzymatic activity, the cyclic nucleotide phosphodiesterase was eluted in good zields with a linear KCl gradient from 0.4 M to 1.8 M. Enzymatic activity determination performed with both cyclic AMP and cyclic GMP as substrate, either at low (0.25 μM) or at high (25 μM) concentration, pointed out the presence of several phosphodiesterase forms with different substrate specificities, in the elution profiles.     

Purification of an insulin-sensitive cyclic AMP phosphodiesterase from rat liver

A low-Km cyclic nucleotide phosphodiesterase solubilised from rat liver membranes by mild proteolysis with chymotrypsin has been purified to apparent homogeneity. The purification included chromatography on cellulose phosphate, Ecteola-cellulose, hydroxyapatite, a theophylline affinity matrix and HPLC on a DEAE-substituted column. The purified enzyme has linear kinetic plots with a Km of 0.24 microM and a Vmax of 6.2 mumol mg-1 min-1 with cyclic AMP as a substrate. It also hydrolyses cyclic GMP with a Km of 0.17 microM and a Vmax which is about a third of that with cyclic AMP. Cyclic GMP is also a competitive inhibitor of cyclic AMP hydrolysis with a Ki of 0.18 microM. The proteolytically solubilised enzyme has a subunit molecular mass of 73 kDa by SDS gel electrophoresis and of 130 kDa by HPLC size-exclusion chromatography, suggesting that it exists as a dimer. A partially purified preparation of this enzyme was used to raise antiserum in a sheep. The antiserum immunoprecipitated activity from liver and adipose tissue of rat and mouse. It had little activity against phosphodiesterase from other rat tissues or other species. Insulin-activated phosphodiesterase from both adipocytes and hepatocytes was immunoprecipitated by the antiserum suggesting that the purified enzyme was an insulin-sensitive phosphodiesterase.     

Identification and characterization of two unusual cGMP-stimulated phoshodiesterases in dictyostelium

Recently, we recognized two genes, gbpA and gbpB, encoding putative cGMP-binding proteins with a Zn(2+)-hydrolase domain and two cyclic nucleotide binding domains. The Zn(2+)-hydrolase domains belong to the superfamily of beta-lactamases, also harboring a small family of class II phosphodiesterases from bacteria and lower eukaryotes. Gene inactivation and overexpression studies demonstrate that gbpA encodes the cGMP-stimulated cGMP-phosphodiesterase that was characterized biochemically previously and was shown to be involved in chemotaxis. cAMP neither activates nor is a substrate of GbpA. The gbpB gene is expressed mainly in the multicellular stage and seems to encode a dual specificity phosphodiesterase with preference for cAMP. The enzyme hydrolyses cAMP approximately 9-fold faster than cGMP and is activated by cAMP and cGMP with a K(A) value of approximately 0.7 and 2.3 microM, respectively. Cells with a deletion of the gbpB gene have increased basal and receptor stimulated cAMP levels and are sporogeneous. We propose that GbpA and GbpB hydrolyze the substrate in the Zn(2+)-hydrolase domain, whereas the cyclic nucleotide binding domains mediate activation. The human cGMP-stimulated cAMP/cGMP phosphodiesterase has similar biochemical properties, but a completely different topology: hydrolysis takes place by a class I catalytic domain and GAF domains mediate cGMP activation.     

Phosphatidylinositol 4,5-bisphosphate phosphodiesterase in higher plants.

A phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate to release inositol trisphosphate was detected in a sedimentable fraction from celery and from some other higher plants. The particulate enzyme also hydrolyses phosphatidylinositol, whereas the soluble phosphatidylinositol phosphodiesterase described previously [Irvine, Letcher & Dawson (1980) Biochem. J. 192, 279-283] acts only on phosphatidylinositol, and we were unable to detect activity of this soluble activity on phosphatidylinositol 4,5-bisphosphate. Activity of the particulate enzyme is markedly enhanced in the presence of deoxycholate, but not of other detergents; the particulate enzyme can also be solubilized by extraction with deoxycholate.     

Preparation of immobilized phosphodiesterase from calf spleen and its use in oligonucleotide analysis

The phosphodiesterase from calf spleen (EC 3.1.4.18) was immobilized on several supports. Some properties of the most suitable enzyme support system--calf spleen phosphodiesterase bound to agarose-Concanavalin A--were investigated, e.g., pH dependence, influence of ionic strength of the buffer medium, and Zn2+-ion inhibition. The immobilized spleen phosphodiesterase showed about 60% of the activity of the free enzyme; the activity toward several oligonucleotide test substrates was unchanged for two months.     

Isolation and partial characterization of a cyclic GMP-dependent cyclic GMP-specific phosphodiesterase from Dictyostelium discoideum

The cellular slime mold, Dictyostelium discoideum, contains at least two classes of phosphodiesterase activity. One class of enzymes hydrolyses cyclic AMP (cAMP) and cyclic GMP (cGMP) with approximately equal rates. Another enzyme, which is less than 5% of the total activity, specifically hydrolyses cGMP. The cGMP-specific enzyme does not bind to a Con A-Sepharose column, while all the cAMP-hydrolyzing activities are retarded by this column. The cGMP-specific enzyme is activated by low cGMP concentrations (10^?8-10^?6 M); the enzyme has normal Michaelis-Menten kinetics at high substrate concentrations with a K_m of about 3–6 μM. The cGMP-binding sites for activation and for catalysis show different cyclic nucleotide specificity, but they are probably located on one protein with a molecular weight of about 70 000. The enzyme is stable only under specific conditions, and the activation property of the enzyme is lost relatively easy. Irreversible modifications occur at temperatures below 0° and above 30°C, and at pH below 6.0. Several other conditions such as high ion concentrations, temperatures just above 0°C and pH above 8.0 lead to reversibel modifications of enzyme activity.     

Synthesis and enzymatic characterization of P1-thio-P2-oxo trideoxynucleoside diphosphates having AZT, FdU, or dT at the 3'-position

Model compounds for oligonucleotide-prodrugs, P1-thio-P2-oxo-trideoxyribonucleoside diphosphates: d[G(s)C(o)X] and d[T(s)A(o)X] (X = AZT, FdU or dT) have been prepared, and their hydrolyses by snake venom phosphodiesterase and nuclease S1 are described.     

Partial purification and properties of a multifunctional 3′,5′-cyclic nucleotide phosphodiesterase from Lactuca cotyledons

The extraction, partial purification and properties of a 3′,5′-cyclic nucleotide phosphodiesterase from lettuce cotyledons is described. Purification involved fractional precipation with (NH₄)₂SO₄, chromatography on Sephadex G-200, affinity chromatography on Affi-Gel Blue and non-denaturing polyacrylamide gel electrophoresis. The behaviour of the final enzyme preparation on SDS-polyacrylamide gel electrophoresis was examined and inidcated an M, of ca 62 000. The enzyme from 3′,5′-cyclic nucleotide phosphodiesterases previously isolated from plant tissues in that it exhibits activity towards pyrimidine as well as purine cyclic nucleotides. Furthermore, it hydrolyses cyclic CMP at a comparable rate to that with which it hydrolyses cyclic AMP and cyclic GMP. Both 3′- and 5′-AMP were released, with the 5′-nucleotide being the major product. Whereas the K_m with all three substrates remained constant during the purification procedure, V_max with cyclic AMP was lower than that for cyclic CMP but increased as purification proceeded. The effects were examined of a range of di- and trivalent metal ions on the enzyme activity. Fe³⁺ significantly stimulated the activity, more so when cyclic GMP was the substrate. Cu²⁺ inhibited the activity.     

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.     

Investigation of the relationship between protein-protein interaction and catalytic activity of a heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) by protein microarray

Ec DOS, a heme-regulated phosphodiesterase from Escherichia coli, is composed of an N-terminal heme-bound PAS domain and a C-terminal phosphodiesterase domain. The heme redox state in the PAS domain regulates Ec DOS phosphodiesterase activity. Interestingly, the isolated heme-bound PAS fragment enhances phosphodiesterase activity of full-length Ec DOS. The enhancement is also regulated by the heme redox state of the isolated PAS domain. In the present study, we used a newly developed protein microarray system to examine the relationship between catalytic activity and the interaction of full-length Ec DOS and the isolated PAS fragment. Adenosine 3',5'-cyclic monophosphate (cAMP), a substrate of the Ec DOS phosphodiesterase, was found to be indispensable for the interaction between Ec DOS and the PAS fragment, and two phosphodiesterase inhibitors, 3-isobutyl-methyl-xanthine and etazolate hydrochloride, hindered the interaction. In addition, an enzyme with a mutation in the putative cAMP-binding sites (H590 and H594) was unable to interact with Ec DOS and lacked enzymatic activity. These results strongly suggest a close relationship between Ec DOS phosphodiesterase activity and interaction with the isolated PAS fragment. Therefore, this study provides insights into the mechanism of how the isolated PAS domain activates Ec DOS, which has important implications for the general role of the isolated PAS domain in cells. Moreover, we found that multiple microscale analyses using the protein microarray system had several advantages over conventional affinity column methods, including the quantity of protein needed, the sensitivity, the variability of immobilized protein, and the time required for the experiment.     

A rapid direct assay of 3'-5' cyclic nucleotide phosphodiesterase activity using chromatography on immobilized acriflavin

A chromatographic method using immobilized acriflavin has been developed for the separation of unreacted cyclic nucleotides from their corresponding 5'-nucleotides, in a direct assay of 3'-5' cyclic nucleotide phosphodiesterase using [3H]-cyclic nucleotides as substrate. The method based on the so-called charge-transfer overlap recognition between flavin and indol rings, provides a rapid (15-20 min) and sensitive elution of [3H]-5'nucleotides with high recovery (up to 98%) and low blanks, while [3H]-cyclic nucleotides are retarded on the column. By this method, the formation of some secondary products by purine metabolizing enzymes such as 5'-nucleotidases, nucleosidases and/or deaminases is taken into account, using [14C]-5'-AMP thus allowing an accurate determination of phosphodiesterase activity in any preparations.     

Studies on the catalytic action of poly-alpha-amino acids. VII. Stereospecificity in the enzyme-like hydrolysis of benzoyl-L-(D)-arginine-p-nitroanilides by copoly (Cys, Glu).

The substrate specificity in the hydrolysis of L-, DL-, and D-BAPA (benzoylarginine-p-nitro-anilide) by copoly (L-Cys, L-Glu) and copoly (D-Cys, D-Glu) was studied, and enzyme-like stereospecific hydrolyses by poly-alpha-amino acids were identified for the first time. The L-type copolymer hydrolyzed L-BAPA faster than D-BAPA and the rates (v) of BAPA hydrolyses by L-type copolymer were found to be in the order vL greater than vDL greater than vD. On the other hand, the D-type copolymer hydrolysed D-BAPA faster than L-BAPA and the rates of BAPA hydrolyses by D-type copolymer were in the order vD greater than vDL greater than vL. In all cases, the reaction followed Michaelis-Menten kinetics when the substrate concentration was corrected, and the optimum conditions of the reaction were pH 6.0 and 40 degrees. The activity appeared after a certain amount of BAPA had combined with the polymer. D- and L-substrates combine competitively with the polymer and the different rates of hydrolysis are presumably due to the different substrate configurations in relation to the conformation of the active site in the polymer. The polymer shows activity near the range of random coil conformation, where some alpha-helical conformation is still present. Only some of the cysteine residues in the copolymer are involved in the hydrolytic activity.     

Inhibition of carboxypeptidase A catalyzed peptide hydrolysis by 3-phenylpropanoate at activating and nonactivating substrate concentrations.

The carboxypeptidase A catalyzed hydrolyses of five structurally related dipeptide substrates in the presence of the inhibitor 3-phenylpropanoate have been studied. At nonactivating substrate concentrations, 3-phenylpropanoate is a mixed inhibitor of carbobenzoxyglycyl-L-phenylalanine hydrolysis and a noncompetitive inhibitor of the hydrolyses of benzoylglycyl-L-phenylalanine, cinnamoylglycyl-L-phenylalanine, hydrocinnamoylglycyl-L-phenylalanine, and acetylglycyl-L-phenylalanine. When carbobenzoxyglycyl-L-phenylalanine and benzoylglycyl-L-phenylalanine exhibit substrate activation, inhibition by 3-phenylpropanoate is mixed but appears to be mostly competitive. Proposed here is a site for the binding of 3-phenylpropanoate along with a kinetic mechanism consistent with these data.     

Synthesis, biophysical, and biochemical properties of PNA-DNA chimeras

Three PNA-DNA chimeric dimer synthons (tT, upT and uhT, see Sch. 1) have been synthesized in solution and used to make T20-analogue chimeras applying standard solid-phase DNA synthesis protocol. Duplex forming ability of chimeras with dA20 and their hydrolyses by 3'- and 5'-exonucleases (snake venom and bovine spleen phosphodiesterase, respectively) have been investigated.     

A family of phosphohydrolases from bovine intestinal mucosa: 5'-nucleotidase

Bovine intestinal 5'-nucleotidase has been partially purified and characterized for comparison with two other phosphohydrolases from the same tissue, alkaline phosphatase and 5'-nucleotide phosphodiesterase, which are closely related structurally and mechanistically. Kinetic studies with a variety of nucleotides and phosphonate analogs show that, although 5'-nucleotidase is a monoesterase like alkaline phosphatase, it more closely resembles 5'-nucleotide phosphodiesterase in its high affinity and specificity for nucleotide binding. 5'-Nucleotidase is bound very strongly by an affinity column containing a bound phosphonate analog of ADP but is not bound by an affinity column containing a non nucleotide phosphonate which selectively binds alkaline phosphatase. 5'-Nucleotidase is strongly bound by immobilized antibodies prepared against 5'-nucleotide phosphodiesterase, and is less strongly bound by immobilized antibodies prepared against alkaline phosphatase. We conclude that 5'-nucleotidase is structurally more similar to 5'-nucleotide phosphodiesterase than to another monoesterase, alkaline phosphatase.     

Cyclic nucleotide phosphodiesterase from a particulate fraction of rat heart. Solubilization and characterization of a single enzymatic form

Approximatively 2–8% of the cyclic nucleotide phosphodiesterase activity of a crude 1000 g supernatant from rat heart was associated with the washed 105,000 g pellet fraction. This activity exhibited biphasic Lineweaver-Burk plots over a large range of cyclic nucleotides concentrations. Concave-Bownward plots were obtained with cyclic AMP as the assay substrate, while cyclic GMP gave rise to concave-upward plots. Treatment of this particulate fraction by freezing and thawing and then with 2% Lubrol PX released the major part of phosphodiesterase activity into the supernatant (70 and 90% for cyclic AMP and cyclic GMP phosphodiesterase activities respectively). Isoelectric focusing of the solubilized enzyme revealed a single peak of phosphodiesterase activity. While the Lineweaver-Burk plots of cyclic AMP phosphodiesterase activity were not markedly modified by detergent treatment kinetic plots of cyclic GMP phosphodiesterase activity underwent a drastic transformation during the overall solubilization procedure. The substantial increase in the cyclic GMP rate of hydrolysis observed at low substrate level might explain the difference in the apparent yield of solubilization between cyclic AMP and cyclic GMP phosphodiesterase activities.     

The stereochemical course of nucleotidyl transfer catalysed by NAD pyrophosphorylase

NAD pyrophosphorylase catalyses nucleotidyl transfer from adenosine (R)-5'-[alpha-17O]triphosphate to nicotinamide mononucleotide with inversion of configuration at the alpha-P giving (S)-[17O]NAD+. The simplest interpretation of this observation is that the adenylyl group is transferred directly from ATP to the co-substrate by an 'in line' mechanism. It is also shown that snake venom phosphodiesterase hydrolyses NAD+ regio-specifically at the adenylyl terminus of the pyrophosphate bond.