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)

The glutamine synthetase (EC 6.3.1.2) from the N2-fixing bacterium Azotobacter vinelandii was purified to homogeneity by heat treatment, ammonium sulfate precipitation and ion-exchange chromatography. The following molecular parameters were determined: molecular weight 640 000, subunit molecular weight 53 000, partial specific volume 0.710 cm3/g, isoelectric point 4.6, amino acid composition. Most of the molecules are composed of 12 identical subunits but active oligomers of other degrees of polymerization, apparently aggregates with 8, 10 and 24 subunits, were also detected to a lesser extent. The enzymatic activity is regulated via adenylylation-deadenylylation cycles: liberation of AMP was detected upon treatment of the adenylylated form with phosphodiesterase along with a change in the catalytic properties. Adenylylation in vivo is specifically induced by high extracellular ammonia levels. The Km values for the Mg2+-dependent formation of glutamine were independent of the degree of adenylylation for glutamate and ATP, but varied for ammonia. Furthermore the catalytic activity is regulated by several nitrogenous feedback inhibitors. The degree of inhibition in some cases was dependent on the substrate concentrations: the sensitivity towards glycine, alanine and serine decreased with a decreasing ammonia level, while the sensitivity towards ADP or AMP increased with a decreasing ATP concentration. Part of the enzyme (about 30%) seems to be attached to the plasma membrane while the main fraction is found in the cytosol.
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PMID:The glutamine synthetase from Azotobacter vinelandii: purification, characterization, regulation and localization. 2 57

Positive selection procedures for mutants of Salmonella typhimurium lacking cyclic 3', 5'7-adenosine monophosphate (cAMP) phosphodiesterase have been devised. The gene (cpd) coding for this enzyme has been located on the chromosome and shown to be 25% co-transducible with metC using phage P22. The mutants have been used to investigate the role of the enzyme in the control of genes whose expression is known to be dependent on cAMP. Significant alterations in the regulation of some but not others of these genes have been observed in these mutants. Mutants lacking the cAMP phosphodiesterase are more sensitive than their parents to a variety of antibiotics that appear to enter the cell through cAMP-dependent transport systems. They grow faster than the wild type on succinate-ammonia-salts, and glucose-proline-salts media and are inhibited by added cAMP on glucose, citrate, or glycerol-ammonia salts media whereas the wild type is unaffected. Neither the growth of Salmonella typhimurium on glycerol or citrate media nor the level of acid hexose phosphatase in the strain is affected by the loss of cAMP phosphodiesterase. In addition, the mutant strains are extremely sensitive to high levels of cAMP. Loss of the cAMP phosphodiesterase in strains unable to synthesize cAMP (adenyl cyclase negative) reduces by 10-fold the requirement for exogenous cAMP for expression of catabolite-sensitive phenotypes. These results suggest that through its control of cAMP levels in the cell the phosphodiesterase may be involved in the regulation of certain classes of catabolite-sensitive operaons and also in protecting the cell against high levels of cAMP.
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PMID:Cyclic 3', 5'-adenosine monophosphate phosphodiesterase mutants of Salmonella typhimurium. 16 78

Derivatives of adenosine 3',5'-cyclic phosphate (cAMP) with modifications in both the 2' and the 8 positions were synthesized and their enzymic activities as activators of cAMP-dependent protein kinase and as substrates for and inhibitors of cAMP phosphodiesterases were determined. Three types of derivatives were investigated: 8-substituted derivatives of O2'-Bt-cAMP, 8-substituted derivatives of 9-beta-D-arabinofuranosyladenine 3',5'-cyclic phosphate (ara-cAMP), and 8-substituted derivatives of 8,2'-anhydro-9-beta-D-arabinofuranosyladenine 3,'5'-cyclic phosphate (8,2'-anhydro-cAMP). The 8-substituted O2'-Bt-cAMP derivatives were synthesized by acylation of the preformed 8-substituted cAMP (8-HS-cAMP, 8-MeS-cAMP, and 8-PhCH2S-cAMP). 8-Br-O2'-tosyl-cAMP was sued as an intermediate for the preparation of 8,2'-anhydro-cAMP derivatives (8-HO-, 8-SH-, 8-H2N-, and 8-H3 CHN derivatives of 8,2'-anhydro-cAMP). 8-Substituted ara-cAMP derivatives were obtained by ring opening of 8-HO-8,2'-anhydro-cAMP with H+/H2O, NH3/MeOH, or MeONa/MeOH (to yield the 8-HO-, 8-H2N-, and 8-MeO-ara-cAMP derivatives). All of these doubly modified derivatives of cAMP are less than one-hundredth as active as cAMP at activating protein kinase and did not serve as substrates for the phosphodiesterase. These data show that the general inactivity of 2' derivatives of cAMP with kinase was not overcome by addition of an 8-substituent, even though many 8-substituted derivatives of cAMP activate the kinase more efficiently than does cAMP itself. In addition they show that while 2'-modification were tolerated by the phosphodiesterase, addition of an 8-substituent countermanded the allowable 2'-modification. The 8-substituted derivates of 02'-Bt-cAMP were found in general to be slightly better inhibitors of phosphodiesterase than the parent compounds containing no o2'-Bt substitution. As a group, the 8-substituted ara-cAMP derivatives were poorer inhibitors of phosphodiesterase than 8-substituted cAMP derivatives while the 8,2'-anhydro-cAMP derivatives were much poorer inhibitors than the 8-substituted ara-cAMP derivatives.
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PMID:8-Substituted derivatives of adenosine 3',5'-cyclic phosphate require an unsubstituted 2'-hydroxyl group in the ribo configuration for biological activity. 17 Sep 58

Cyclic-AMP and ammonia have been previously identified as extracellular signals during Dictyostelium development. Both are important in controlling morphological movements and cyclic-AMP also in inducing gene expression. The work in tis paper is concerned with their effects on developmental gene expression. Cyclic-AMP was found to act as an inducer during the aggregative (as exemplified by phosphodiesterase) and the post-aggregative (glycogen phosphorylase, UDP-galactose polysaccharide transferase, prespore vacuoles and stalk cells) phases of gene expression. Ammonia inhibited the appearance of each of the above markers and antagonized the inductive effects of cyclic-AMP on them. This inhibition by ammonia of cyclic-AMP inducible gene expression may involve a step linking elevated intracellular cyclic-AMP levels to gene activation. It has been suggested that the specification of cells within the aggregate into the stalk and spore pathways of differentiation might be controlled by cyclic-AMP and ammonia. In this model for pattern formation cyclic-AMP would induce stalk cell differentiation and ammonia spore formation. The present results argue against this idea since cyclic-AMP induces and ammonia inhibits differentiation along both pathways. The function of these agents may rather be to coordinate the rates of biochemical differentiation of individual cells and link them to the overall morphological changes occurring during development.
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PMID:Gene expression in Dictyostelium discoidium: mutually antagonistic roles of cyclic-AMP and ammonia. 23 Feb 81

We examined expression of the Dictyostelium cAMP phosphodiesterase (PDE) gene under conditions that alter intracellular cAMP levels during in vitro differentiation of wild-type strain V12M2 and a sporogenous derivative, HB200. In control cultures, cellular PDE activity peaked at 6 hr and declined by 8 hr, while secreted PDE activity continued to increase through 8 hr. Lowering intracellular cAMP levels with caffeine or progesterone increased cellular and secreted PDE activities 2-fold, increased stalk cell differentiation, and inhibited spore differentiation. In contrast, exposure to 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP; a membrane-permeable cAMP analog) or ammonia (which promotes intracellular cAMP accumulation in V12M2 and HB200 cells) lowered PDE activities by as much as 45%, decreased stalk cell differentiation, and increased spore differentiation. Simultaneous exposure to 8-Br-cAMP and caffeine gave intermediate PDE activities as would be expected if 8-Br-cAMP entered the cell and bypassed the caffeine-mediated block to adenylate cyclase activation. In all cases, we observed commensurate changes in developmental PDE transcript levels. The developmental time course of expression was not significantly altered by these treatments. These results suggest that the magnitude of PDE gene expression is negatively regulated by intracellular cAMP levels and provide evidence for one of the earliest changes in gene expression that is consistent with cell-type specificity. These results are discussed in terms of a bistable switch employing intracellular cAMP as a regulator of cell fate.
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PMID:Conditions that alter intracellular cAMP levels affect expression of the cAMP phosphodiesterase gene in Dictyostelium. 216 56

The role of cyclic nucleotides as intracellular second messengers mediating the excitatory chronotropic and inotropic actions of octopamine (OCT) and dopamine (DA) on the neurogenic Limulus heart was investigated. Tissue levels of cAMP, but not cGMP, were significantly increased in isolated cardiac ganglia and cardiac muscle following 10 min exposure to 10(-5) M OCT or 10(-5) M DA. In both tissues, OCT elicited larger increases in cAMP than did DA. Amine-induced cAMP accumulation in the cardiac ganglion and in the cardiac muscle was prevented by the alpha-adrenergic blocker phentolamine. The adenylate cyclase activator forskolin and the phosphodiesterase inhibitor IBMX produced amine-like chronotropic and inotropic effects when applied to the isolated heart preparation. However, the kinetics of the responses differed for the two agents. Additional pharmacological agents (RO-20-1724, papaverine, SQ 20,009, and 8-parachloro-phenylthio cAMP) also had amine-like effects but to a lesser extent. The chronotropic, but not inotropic, effects of OCT and DA were potentiated in the presence of IBMX. These data suggest that a cAMP-dependent mechanism underlies the excitatory effects of the neuromodulators OCT and DA on the Limulus heart.
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PMID:Mechanism for amine modulation of the neurogenic Limulus heart: evidence for involvement of cAMP. 244 16

The modification of Klenow fragment of DNA polymerase I E. coli was investigated by the affinity reagents d(Tp)2C[Pt2+(NH3)2OH](pT)7 and d(pT)2pC[Pt2+(NH3)2OH](pT)7. The template binding site of the enzyme was modified by these reagents in the presence of NaF (5 mM), which inhibits selectively the 3'----5'-exonuclease activity of the enzyme and therefore prevents the reagent from degradation. NaCN destroyed covalent bonds between reagents and enzyme, restoring activity of the Klenow fragment. The affinity of different ligands (inorganic phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of Klenow fragment was estimated. Minimal ligands capable to bind with the template site were shown to be triethylphosphate (Kd 290 microM) and phosphate (Kd 26 microM). Ligand affinity increases by the factor 1.76 per an added (monomer unit from phosphate to d(pT) and then for oligonucleotides d(Tp)nT (n 1 to 19-20). At n greater than 19-20, the ligand affinity remained constant. The complete ethylation of phosphodiester groups lowers affinity of the oligothymidylates to the enzyme by approximately 10 times, and comparable decrease of Pt2+-oligonucleotide affinity to polymerase is caused by the absence of Mn2+-ions. The data obtained led to suggestion that one Me2+-dependent electrostatic contact of the template phosphodiester group with the enzyme takes place (delta G = -1.45...-1.75 kcal/mole). Formation of a hydrogen bond with the oxygen atom of P = O group of the same template phosphate is also assumed (delta G = -4.8...-4.9 kcal/mole). Other template internucleotide phosphates do not interact with the enzyme but the bases of oligonucleotides take part in hydrophobic interactions with the template binding site. Gibbs energy changes by -0.34 kcal/mole when the template is lengthened by one unit.
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PMID:[Klenow fragment of DNA-polymerase I from E. coli. III. The role of internucleotide phosphate groups of the matrix in its binding with the enzyme]. 266 77

Mycobacterium avium was previously shown to be dependent upon ammonia or glutamine as a nitrogen source. In an effort to assess the physiology of ammonia assimilation by M. avium, a characterization of its glutamine synthetase was performed. The enzyme from M. avium was purified by streptomycin sulfate treatment, ammonium sulfate precipitation, and affinity chromatography. The enzyme was unusual in that it had a pH optimum of 6.4 and maximum enzyme activity was obtained between 50 and 60 degrees C as shown by the transferase assay. The glutamine synthetase activity from batch-cultured cells decreased with increasing concentration of ammonium chloride in the range of 0.25-5 mumol/mL of medium, which demonstrated a response to environmental supply of a nitrogen source. The mycobacterial enzyme was similar to the other bacterial glutamine synthetases in terms of molecular weight and sedimentation coefficient which were 600 000 and 19.5 S, respectively, and enzyme activity was lost by treatment with a glutamate analog, methionine sulfoximine. The isoelectric point was, however, pH 4.5. Treatment of the enzyme with snake venom phosphodiesterase resulted in an increase in specific activity. AMP was released by the phosphodiesterase treatment, thus demonstrating that M. avium glutamine synthetase was regulated by adenylylation modification.
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PMID:Glutamine synthetase from Mycobacterium avium. 614 81

NAD kinase, one of the first enzymes activated after fertilization of sea urchin eggs, is regulated by Ca2+ and calmodulin in vitro. The evidence is the requirement for low amounts of Ca2+ (Kd for Ca2+ of 4 x 10(-7) M) and the dissociation of a heat-stable activator from the enzyme which is similar to calmodulin on the basis of radioimmunoassay, activation of bovine brain phosphodiesterase and coelectrophoresis of a major protein of the activator fraction with bovine calmodulin. Also, the calcium stimulation of the enzyme is prevented by trifluoperazine, an inhibitor of calmodulin-associated reactions. In vivo studies show that the enzyme is activated by artificial parthenogenesis regimes that increase cytosolic Ca2+, but not by ammonia activation which only partially activates eggs and bypasses the Ca2+-rise step. These in vitro and in vivo studies indicate that calmodulin is part of the linkage between the rise in Ca2+ at fertilization and the turning on of egg metabolism.
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PMID:Calmodulin activates NAD kinase of sea urchin eggs: an early event of fertilization. 625 5

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5'-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5'-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5'-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5'-phosphoramidate. The enzyme that catalyses the formation of adenosine 5'-phosphoramidate from ammonia and adenosine 5'-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH(4))(2)SO(4) precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2-agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000-65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3'-phosphate 5'-phosphosulphate will not replace adenosine 5'-phosphosulphate, and the apparent K(m) for the last-mentioned compound is 0.82mm. The apparent K(m) for ammonia (assuming NH(3) to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5'-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5'-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5'-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5'-phosphosulphate kinase, adenosine 5'-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5'-phosphoramidate is found in old samples of the ammonium salt of adenosine 5'-phosphosulphate and can be formed non-enzymically if adenosine 5'-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5'-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5'-phosphoramidate by selectively speeding up an already favoured reaction.
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PMID:Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5' -phosphoramidate from adenosine 5' -phosphosulphate and ammonia. 627 7


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