Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel phosphodiesterase was purified from cultured tobacco cells to a state which appeared homogeneous on polyacrylamide gel electrophoresis. The enzyme hydrolyzed various phosphodiester and pyrophosphate bonds, including p-nitrophenyl thymidine 5'-phosphate, p-nitrophenyl thymidine 3'-phosphate, cyclic nucleotides, ATP, NAD+, inorganic pyrophosphate, dinucleotides, and poly(adenosine diphosphate ribose), which is a polymer synthesized from NAD+. However, it did not hydrolyze highly polymerized polynucleotides. The molecular weight of the native enzyme was estimated as 270 000 to 280 000 by gel filtration on Sephadex G-200 and Bio-Gel A-5m. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was composed of subunits with molecular weights calculated to be 75 000. The enzyme did not require divalent cations for activity being fully active in the presence of ethylenediaminetetraacetic acid. The pH optimum for the enzyme was approximately 6 with p-ni-trophenyl thymidine 5'-phosphate or adenosine cyclic 3',5'monophosphate, and 5.3 with NAD+. Double reciprocal plots of the initial velocity against the concentration of p-nitrophenyl thymidine 5'-phosphate gave two apparent Km values of 0.17 and 1.3 mM, suggesting the presence of at least two active sites.
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PMID:A novel phosphodiesterase from cultured tobacco cells. 0 41

Transducin (T alpha beta gamma), the heterotrimeric GTP-binding protein that interacts with photoexcited rhodopsin (Rh*) and the cGMP-phosphodiesterase (PDE) in retinal rod cells, is sensitive to cholera (CTx) and pertussis toxins (PTx), which catalyze the binding of an ADP-ribose to the alpha subunit at Arg174 and Cys347, respectively. These two types of ADP-ribosylations are investigated with transducin in vitro or with reconstituted retinal rod outer-segment membranes. Several functional perturbations inflicted on T alpha by the resulting covalent modifications are studied such as: the binding of T alpha to T beta gamma to the membrane and to Rh*; the spontaneous or Rh*-catalysed exchange of GDP for GTP or guanosine 5-[gamma-thio]triphosphate (GTP[gamma S]), the conformational switch and activation undergone by transducin upon this exchange, the activation of T alpha GDP by fluoride complexes and the activation of the PDE by T alpha GTP. ADP-ribosylation of transducin by CTx requires the GTP-dependent activation of ADP-ribosylation factors (ARF), takes place only on the high-affinity, nucleotide-free complex, Rh*-T alpha empty-T beta gamma and does not activate T alpha. Subsequent to CTx-catalyzed ADP-ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive CTxT alpha GDP (CTxT alpha, ADP-ribosylated alpha subunit of transducin) which remains associated to T beta gamma; (b) CTxT alpha GDP-T beta gamma exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP[gamma S] in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as gamma-phosphate analogs, are kinetically modified, suggesting that the ADP-ribose on Arg174 specifically perturbs binding of the gamma-phosphate in the nucleotide site; (c) CTxT alpha GDP-T beta gamma can still couple to Rh* and undergo fast nucleotide exchange; (d) CTxT alpha GTP[gamma S] and CTxT alpha GDP-AlFx (AlFx, Aluminofluoride complex) activate retinal cGMP-phosphodiesterase (PDE) with the same efficiency as their unmodified counterparts, but the kinetics and affinities of fluoride activation are changed; (e) CTxT alpha GTP hydrolyses GTP more slowly than unmodified T alpha GTP, which entirely accounts for the prolonged action of CTxT alpha GTP on the PDE; (f) after GTP hydrolysis, CTxT alpha GDP reassociates to T beta gamma and becomes inactive. Thus, CTx catalyzed ADP-ribosylation only perturbs in T alpha the GTP-binding domain, but not the conformational switch nor the domains of contact with the T beta gamma subunit, with Rh* and with the PDE.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Functional modifications of transducin induced by cholera or pertussis-toxin-catalyzed ADP-ribosylation. 133 64

When the homogenate prepared from immature rat testes was incubated with [32P]NAD, several proteins (90, 39 and 20 kDa) were ADP-ribosylated in the absence of bacterial toxins. This observation suggested the existence of an endogenous ADP-ribosyltransferase and substrates. The data that the digested product by phosphodiesterase of ADP-ribosylated 20 kDa protein was 5'-AMP suggested that 20 kDa protein was mono(ADP-ribosyl)ated. In addition, the mono(ADP-ribosyl)ation of 20 kDa protein was enhanced by guanine nucleotides such as GTP, GDP and GTP[gamma S], and decreased by the concentrations of 10 mM Mg2+. In contrast, the incorporation of ADP-ribose moiety from NAD to both 90 and 39 kDa proteins was not changed by guanine nucleotides. On the other hand, mono(ADP-ribosyl)ation of 20 kDa protein was not observed in the homogenate prepared from other tissues of the same rats. Furthermore, we found that mono(ADP-ribosyl)ation of 20 kDa protein was decreased with the maturation of the rats and that an endogenous mono(ADP-ribosyl)transferase and 20 kDa protein were located in the nuclei.
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PMID:Reduction of mono(ADP-ribosyl)ation of 20 kDa protein with maturation in rat testis: involvement of guanine nucleotides. 189 5

The possible role of adenosine 3',5'-cyclic monophosphate (cAMP) in the mechanism of the acute inhibitory effects of nicotinamide and analogues on brush-border membrane (BBM) phosphate transport was investigated. Compared with basal values, cAMP content of rat renal proximal tubule suspensions was elevated two- to fivefold when incubated at 37 degrees C for 1 h with nicotinamide, 5-methylnicotinamide, or picolinamide at 1-3 mM and in the presence of a phosphodiesterase inhibitor. Thymidine had no effect on cAMP content. There was significant and specific inhibition of BBM transport of phosphate when proximal tubules were incubated with either nicotinamide or picolinamide at concentrations that increased tubule cAMP content. Thymidine had no effect on BBM transport of phosphate. These findings were independent of the dietary Pi intake of the rats. The absence of any effect of thymidine on phosphate transport strongly suggests that inhibition of poly(adenosine diphosphate ribose) polymerase does not play a role in nicotinamide action on phosphate transport. The change in phosphate transport induced by nicotinamide occurred with no change in NAD content. These findings indicate that an increase in cAMP, rather than NAD, is the important change that may mediate the acute inhibition of Na(+)-dependent phosphate transport by nicotinamide.
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PMID:Increased cAMP in proximal tubules is acute effect of nicotinamide analogues. 255 65

Binding of polyvalent antigens to IgE present in Fc epsilon receptors on the surface of mast cells and the RBL-2H3 cell line triggers the exocytotic release of allergic mediators. Preincubation of RBL-2H3 cells with cholera toxin (CT) was found to potentiate greater than or equal to 2- to 3-fold the rate and final amount of antigen-induced secretion of [3H]serotonin and N-acetyl beta-D-glucosaminidase. This was accompanied by a more variable increase in the initial rate of antigen-triggered formation of inositol phosphates. The holotoxin was required for potentiation, as neither the A nor the B subunit was effective when added separately. Four observations indicate that cAMP was not the primary effector of the augmentation of secretion caused by CT: (i) culture conditions were found in which CT caused large increases in secretion but very modest (or no) increases in cAMP; (ii) under other conditions, progressive increase in [CT] caused a maximum 2.5- to 3-fold increase in cAMP followed by a return to basal levels, whereas the secretory response saturated and remained stable; (iii) permeant cAMP analogs consistently enhanced secretion at low doses and inhibited at higher doses, but the peak enhancement was always much less than that achieved by an optimal dose of CT; (iv) the selective phosphodiesterase inhibitor Ro 20-1724 exhibited similar biphasic dose-response curves, the maximum enhancement again being small compared to that caused by CT itself. Both in vitro and in vivo, CT catalyzed transfer of ADP-ribose from NAD to two membrane proteins that comigrated on NaDodSO4/polyacrylamide gel electrophoresis with two CT substrates in other cell types, and these were identified by immunoblotting as Gs alpha. These results suggest that ADP-ribosylation of a cholera toxin substrate potentiates IgE-mediated secretion from RBL-2H3 cells by a largely cAMP-independent route.
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PMID:Cholera toxin potentiates IgE-coupled inositol phospholipid hydrolysis and mediator secretion by RBL-2H3 cells. 284 4

In order to exert its antitumor effects, the C-nucleoside tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is converted to the dinucleotide TAD (thiazole-4-carboxamide adenine dinucleotide), an inhibitor of IMP dehydrogenase (IMPD). With few exceptions, sensitive tumors (such as the P388 leukemia) have been found to accumulate substantially more of this inhibitory dinucleotide than resistant strains (exemplified by the colon 38 carcinoma). Previous studies have attributed this difference to a depressed capacity to synthesize TAD on the part of tumors refractory to tiazofurin. In the present study, a second contributory factor has been identified, viz. an enhanced ability to degrade preformed TAD. This degradation has been traced to a soluble phosphodiesterase present at high levels in tumors naturally resistant to tiazofurin. Using standard techniques, this TAD-phosphodiesterase has been purified 200-fold from the colon 38 carcinoma. The activity so purified readily hydrolyzed TAD and ADP-ribose, but exhibited a comparatively weak activity toward NAD and thymidine-5'-monophosphate-nitrophenyl ester. ADP-Ribose was also an excellent inhibitor of the hydrolysis of TAD. It is concluded, on the basis of these results, that TAD-phosphodiesterase plays an important role in the expression of the oncolytic activity of tiazofurin. The suggestion is also made that ADP-ribose may be the natural substrate for this enzyme.
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PMID:Studies on the mechanism of action of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide). VI. Biochemical and pharmacological studies on the degradation of thiazole-4-carboxamide adenine dinucleotide (TAD). 287 71

Separated macromolecular fractions of in vitro synthesized poly(ADP-ribose) by liver nuclei were subjected to ion-exchange chromatography in a programmed high-performance liquid chromatographic elution system. The effects of ionic strength, pH and temperature on the separation of poly(ADP-ribose) chains were determined. Short chain oligomers (up to n = 11) were fractionated into individual components by baseline separation. Each fraction was analyzed for chain length. Trace amounts of Ado(P)Rib(P)Rib(P) found in phosphodiesterase digests were taken as indication of apparent branching. In phosphodiesterase digests of the shorter oligomers, besides traces of the above component, two other digestion products were also observed, presumably representing oligomer termini, one terminal fragment being dominant in short oligomers. Medium and long chain oligomers were partly resolved to individual components, and especially the long oligomers exhibited marked temperature dependent elution patterns. Apparent branching increased with increasing chain length up to about 3% for n = 44 and components presumably indicating termini diminished to mere traces. The adenine spectra of all fractions identified individual components.
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PMID:High-performance liquid chromatography of in vitro synthesized poly(ADP-ribose) on ion-exchange columns, separation of oligomers of varying chain length and estimation of apparent branching. 299 69

Thiols such as cysteine and dithiothreitol are substrates for the ADP-ribosyltransferase activity of pertussis toxin. When cysteine was incubated with NAD+ and toxin at pH 7.5, a product containing ADP-ribose and cysteine (presumably ADP-ribosylcysteine) was isolated by high-performance liquid chromatography, and characterized by its composition and release of AMP with phosphodiesterase. Cysteine has a Km of 105 mM at saturating NAD+ concentration. The ability of thiols to act as a substrate is one explanation for the very high concentrations (250 mM or greater) that have been observed to enhance the apparent NAD glycohydrolase activity of the toxin.
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PMID:Thiol reagents are substrates for the ADP-ribosyltransferase activity of pertussis toxin. 313 46

Adenyl-32P-Labeled 3'-deoxy-NAD+ was utilized as a substrate by pure DNA-dependent poly(ADP-ribose)polymerase (EC 2.4.2.30) from calf thymus in the automodification reaction with an apparent Km of 20 microM and a Vmax of 80 nmol/min/mg of protein. Analysis by lithium lauryl sulfate-polyacrylamide gel electrophoresis revealed a single 32P-labeled protein of 116-kDa which comigrated with automodified enzyme. Addition of increasing amounts of histone H1 up to a concentration of 15 micrograms/ml stimulated the synthesis of protein-bound polymers of 3'-deoxy-ADP-ribose. However, the average polymer size was equal to 2 in the presence and 4 in the absence of histone H1, respectively. The synthesis of protein-bound oligomers of 3'-deoxy-ADP-ribose was inhibited by the polymerase inhibitors benzamide, nicotinamide, thymidine, and NaCl. A pulse labeling of polymer synthesis with 40 microM [32P]3'-deoxy-NAD+ either in the presence or absence of 15 micrograms/ml of histone H1, followed by a chase with 1 mM [3H]NAD+, was used to determine the mechanism of poly(ADP-ribose) elongation. Following enzyme digestion of these polymers with phosphodiesterase, it was found that 52 and 24% of the total 32P radiolabel was associated with the 3'-deoxy-AMP termini of the polymers synthesized in the pulse reactions, in the presence or absence of histone H1, respectively. In contrast, less than 10% of the total radioactivity was associated with 3'-deoxy-AMP in the product of the chase reactions. These results are consistent with the conclusion that the initially attached residue of 3'-deoxy-ADP-ribose to either the polymerase or histone H1, is elongated by the "protein-distal" addition of ADP-ribose residues to the AMP terminus of the growing polymer chain.
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PMID:3'-Deoxy-NAD+ as a substrate for poly(ADP-ribose)polymerase and the reaction mechanism of poly(ADP-ribose) elongation. 314 24

2',5'-Oligo(A)synthetase (2-5A) and 2-phosphodiesterase were found in the L cells nuclei. In the cell nuclei 2-5A is 10-30 times higher, than in the cytoplasm. It is induced by interferon and depends on the cell growth state. 2-Phosphodiesterase activity has two pH optima of hydrolysis of 2-5A, namely 7.1, and 7.9 and decreases after interferon treatment of cells. Thus, interferon treatment of cells leads to an increase of the 2-5A level in cell nuclei. One of the possible pathways for 2-5A action in cell nuclei is the regulation of (ADP-ribose)transferase activity. Treatment of L cells with 2-5A (A2pA2pA) leads to activation of ADP-ribosylation of proteins by a factor of 1.5 in a concentration range of 10(-9)-10(-7) M, but more higher concentrations of 2-5A inhibit this process up to 60%. Treatment of cells with actinomycin D has no influence on 2-5A induced changes in protein ADP-ribosylation. This result is indicative for a new pathway of interferon action and 2-5A mediated regulation of cell metabolism.
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PMID:[Metabolism of oligoadenylates in cell nuclei and regulation of protein ADP-ribosylation]. 377 90


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