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Sample records for adenylating enzyme mbta

  1. Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

    PubMed Central

    Duckworth, Benjamin P.; Nelson, Kathryn M.; Aldrich, Courtney C.

    2013-01-01

    Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases. PMID:22283817

  2. Post-translational Acetylation of MbtA Modulates Mycobacterial Siderophore Biosynthesis.

    PubMed

    Vergnolle, Olivia; Xu, Hua; Tufariello, JoAnn M; Favrot, Lorenza; Malek, Adel A; Jacobs, William R; Blanchard, John S

    2016-10-14

    Iron is an essential element for life, but its soluble form is scarce in the environment and is rarer in the human body. Mtb (Mycobacterium tuberculosis) produces two aryl-capped siderophores, mycobactin (MBT) and carboxymycobactin (cMBT), to chelate intracellular iron. The adenylating enzyme MbtA catalyzes the first step of mycobactin biosynthesis in two half-reactions: activation of the salicylic acid as an acyl-adenylate and ligation onto the acyl carrier protein (ACP) domain of MbtB to form covalently salicylated MbtB-ACP. We report the first apo-MbtA structure from Mycobacterium smegmatis at 2.3 Å. We demonstrate here that MbtA activity can be reversibly, post-translationally regulated by acetylation. Indeed the mycobacterial Pat (protein lysine acetyltransferase), Rv0998, specifically acetylates MbtA on lysine 546, in a cAMP-dependent manner, leading to enzyme inhibition. MbtA acetylation can be reversed by the NAD(+)-dependent DAc (deacetyltransferase), Rv1151c. Deletion of Pat and DAc genes in Mtb revealed distinct phenotypes for strains lacking one or the other gene at low pH and limiting iron conditions. This study establishes a direct connection between the reversible acetylation system Pat/DAc and the ability of Mtb to adapt in limited iron conditions, which is critical for mycobacterial infection. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. The crystal structure of the adenylation enzyme VinN reveals a unique β-amino acid recognition mechanism.

    PubMed

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-11-07

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp(230) residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes.

  4. The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism*

    PubMed Central

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-01-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp230 residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  5. Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme.

    PubMed Central

    Rogel, A; Schultz, J E; Brownlie, R M; Coote, J G; Parton, R; Hanski, E

    1989-01-01

    Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis. Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP. This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over-produced both enzymatic and toxic activities of the enzyme. Western blot analysis of the extract using anti-B.pertussis AC antibodies detected only one protein of 200 kd. However, gel filtration of the extract resolved two peaks of enzymatic activity. The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity. Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties. Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP. B.pertussis AC gene expressed in Escherichia coli produced a calmodulin-dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity. It is proposed that a post-translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties. Images PMID:2555185

  6. Colorimetric determination of pyrophosphate anion and its application to adenylation enzyme assay.

    PubMed

    Katano, Hajime; Watanabe, Hiro; Takakuwa, Masahiro; Maruyama, Chitose; Hamano, Yoshimitsu

    2013-01-01

    A colorimetric pyrophosphate assay based on the formation and reduction of the 18-molybdopyrophosphate ([(P2O7)Mo18O54](4-)) anion in an acetonitrile-water mixed solvent was modified and improved. The [(P2O7)Mo18O54](4-) anion is precipitated from the acetonitrile-water solution containing MoO4(2-) and HCl, and is re-dissolved in neat acetonitrile or propylene carbonate. This separation process decreases the interference by ATP, and prevents a yellow coloration of the reducing agent, ascorbic acid, due to excess Mo(VI) species. In the organic solvent, the [(P2O7)Mo18O54](4-) anion is reduced to a more intense blue molybdopyrophosphate species. The application of the colorimetry to the assay of adenylation enzymes is also described in this note.

  7. Metabolic compensation for profound erythrocyte adenylate kinase deficiency. A hereditary enzyme defect without hemolytic anemia.

    PubMed Central

    Beutler, E; Carson, D; Dannawi, H; Forman, L; Kuhl, W; West, C; Westwood, B

    1983-01-01

    A child with hemolytic anemia was found to have severe erythrocyte adenylate kinase (AK) deficiency, but an equally enzyme-deficient sibling had no evidence of hemolysis. No residual enzyme activity was found in erythrocytes by spectrophotometric methods that could easily have detected 0.1% of normal activity. However, concentrated hemolysates were shown to have the capacity to generate small amounts of ATP and AMP from ADP after prolonged incubation. Hemolysates could also catalyze the transfer of labeled gamma-phosphate from ATP to ADP. Intact erythrocytes were able to transfer phosphate from the gamma-position of ATP to the beta-position, albeit at a rate substantially slower than normal. They could also incorporate 14C-labeled adenine into ADP and ATP. Thus, a small amount of residual AK-like activity representing about 1/2,000 of the activity normally present could be documented in the deficient erythrocytes. The residual activity was not inhibited by N-ethylmaleimide, which completely abolishes the activity of the normal AK1 isozyme of erythrocytes. The minute amount of residual activity in erythrocytes could represent a small amount of the AK2 isozyme, which has not been thought to be present in erythrocytes, or the activity of erythrocyte guanylate kinase with AMP substituting as substrate for GMP. Peripheral blood leukocytes, cultured skin fibroblasts, and transformed lymphoblasts from the deficient subject manifested about 17, 24, and 74%, respectively, of the activity of the concurrent controls. This residual activity is consistent with the existence of genetically independent AK isozyme, AK2, which is known to exist in these tissues. The cause of hemolysis in the proband was not identified. Possibilities include an unrelated enzyme deficiency or other erythrocyte enzyme defect and intraction of another unidentified defect with AK deficiency. PMID:6308059

  8. Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

    PubMed

    Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

    2014-11-01

    DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

  9. Understanding thermal adaptation of enzymes through the multistate rational design and stability prediction of 100 adenylate kinases.

    PubMed

    Howell, Stanley C; Inampudi, Krishna Kishore; Bean, Doyle P; Wilson, Corey J

    2014-02-04

    Careful balance between structural stability and flexibility is a hallmark of enzymatic function, and temperature can affect both properties. Canonical (fixed-backbone) enzyme design strategies currently do not consider the role of these properties. Herein, we describe the rational design of 100 temperature-adapted adenylate kinase enzymes using a multistate design strategy that incorporates the impact of conformational changes to backbone structure and stability, in addition to experimental analysis of thermostability and function. Comparison of the experimental temperature of maximum activity to the melting temperature across all 100 variants reveals a strong correlation between these two parameters. In turn, experimental stability data were used to produce accurate predictions of thermostability, providing the requisite complement for de novo temperature-adapted enzyme design. In principle, this level of design-based analysis can be applied to any protein, paving the way toward identifying and understanding the hallmarks of the thermodynamic and structural limits of function.

  10. Design, synthesis, and biological evaluation of α-hydroxyacyl-AMS inhibitors of amino acid adenylation enzymes.

    PubMed

    Davis, Tony D; Mohandas, Poornima; Chiriac, Maria I; Bythrow, Glennon V; Quadri, Luis E N; Tan, Derek S

    2016-11-01

    Biosynthesis of bacterial natural-product virulence factors is emerging as a promising antibiotic target. Many such natural products are produced by nonribosomal peptide synthetases (NRPS) from amino acid precursors. To develop selective inhibitors of these pathways, we have previously described aminoacyl-AMS (sulfamoyladenosine) macrocycles that inhibit NRPS amino acid adenylation domains but not mechanistically-related aminoacyl-tRNA synthetases. To improve the cell permeability of these inhibitors, we explore herein replacement of the α-amino group with an α-hydroxy group. In both macrocycles and corresponding linear congeners, this leads to decreased biochemical inhibition of the cysteine adenylation domain of the Yersina pestis siderophore synthetase HMWP2, which we attribute to loss of an electrostatic interaction with a conserved active-site aspartate. However, inhibitory activity can be regained by installing a cognate β-thiol moiety in the linear series. This provides a path forward to develop selective, cell-penetrant inhibitors of the biosynthesis of virulence factors to probe their biological functions and potential as therapeutic targets.

  11. Synthesis of Chromone, Quinolone, and Benzoxazinone Sulfonamide Nucleosides as Conformationally Constrained Inhibitors of Adenylating Enzymes Required for Siderophore Biosynthesis

    PubMed Central

    Engelhart, Curtis A.; Aldrich, Courtney C.

    2013-01-01

    MbtA catalyzes the first committed step of mycobactin biosynthesis in Mycobacterium tuberculosis (Mtb) and is responsible for the incorporation of salicylic acid into the mycobactin siderophores. 5′-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS) is an extremely potent nucleoside inhibitor of MbtA that possesses excellent activity against whole-cell Mtb, but suffers from poor bioavailability. In an effort to improve the bioavailability, we have designed four conformationally constrained analogues of Sal-AMS that remove two rotatable bonds and the ionized sulfamate group based on computational and structural studies. Herein we describe the synthesis, biochemical, and microbiological evaluation of chromone-, quinolone-, and benzoxazinone-3-sulfonamide derivatives of Sal-AMS. We developed new chemistry to assemble these three heterocycles from common β-ketosulfonamide intermediates. The synthesis of the chromone- and quinolone-3-sulfonamide intermediates features formylation of a β-ketosulfonamide employing dimethylformamide dimethyl acetal to afford an enaminone that can react intramolecularly with a phenol or intermolecularly with a primary amine via addition-elimination reaction(s). The benzoxazinone-3-sulfonamide was prepared by nitrosation of a β-ketosulfonamide followed by intramolecular nucleophilic aromatic substitution. Mitsunobu coupling of these bicyclic sulfonamides with a protected adenosine derivative followed by global deprotection provides a concise synthesis of the respective inhibitors. PMID:23805993

  12. Synthesis of chromone, quinolone, and benzoxazinone sulfonamide nucleosides as conformationally constrained inhibitors of adenylating enzymes required for siderophore biosynthesis.

    PubMed

    Engelhart, Curtis A; Aldrich, Courtney C

    2013-08-02

    MbtA catalyzes the first committed step of mycobactin biosynthesis in Mycobacterium tuberculosis (Mtb) and is responsible for the incorporation of salicylic acid into the mycobactin siderophores. 5'-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS) is an extremely potent nucleoside inhibitor of MbtA that possesses excellent activity against whole-cell Mtb but suffers from poor bioavailability. In an effort to improve the bioavailability, we have designed four conformationally constrained analogues of Sal-AMS that remove two rotatable bonds and the ionized sulfamate group on the basis of computational and structural studies. Herein we describe the synthesis, biochemical, and microbiological evaluation of chromone-, quinolone-, and benzoxazinone-3-sulfonamide derivatives of Sal-AMS. We developed new chemistry to assemble these three heterocycles from common β-ketosulfonamide intermediates. The synthesis of the chromone- and quinolone-3-sulfonamide intermediates features formylation of a β-ketosulfonamide employing dimethylformamide dimethyl acetal to afford an enaminone that can react intramolecularly with a phenol or intermolecularly with a primary amine via addition-elimination reaction(s). The benzoxazinone-3-sulfonamide was prepared by nitrosation of a β-ketosulfonamide followed by intramolecular nucleophilic aromatic substitution. Mitsunobu coupling of these bicyclic sulfonamides with a protected adenosine derivative followed by global deprotection provides a concise synthesis of the respective inhibitors.

  13. Event detection and sub-state discovery from biomolecular simulations using higher-order statistics: application to enzyme adenylate kinase.

    PubMed

    Ramanathan, Arvind; Savol, Andrej J; Agarwal, Pratul K; Chennubhotla, Chakra S

    2012-11-01

    Biomolecular simulations at millisecond and longer time-scales can provide vital insights into functional mechanisms. Because post-simulation analyses of such large trajectory datasets can be a limiting factor in obtaining biological insights, there is an emerging need to identify key dynamical events and relating these events to the biological function online, that is, as simulations are progressing. Recently, we have introduced a novel computational technique, quasi-anharmonic analysis (QAA) (Ramanathan et al., PLoS One 2011;6:e15827), for partitioning the conformational landscape into a hierarchy of functionally relevant sub-states. The unique capabilities of QAA are enabled by exploiting anharmonicity in the form of fourth-order statistics for characterizing atomic fluctuations. In this article, we extend QAA for analyzing long time-scale simulations online. In particular, we present HOST4MD--a higher-order statistical toolbox for molecular dynamics simulations, which (1) identifies key dynamical events as simulations are in progress, (2) explores potential sub-states, and (3) identifies conformational transitions that enable the protein to access those sub-states. We demonstrate HOST4MD on microsecond timescale simulations of the enzyme adenylate kinase in its apo state. HOST4MD identifies several conformational events in these simulations, revealing how the intrinsic coupling between the three subdomains (LID, CORE, and NMP) changes during the simulations. Further, it also identifies an inherent asymmetry in the opening/closing of the two binding sites. We anticipate that HOST4MD will provide a powerful and extensible framework for detecting biophysically relevant conformational coordinates from long time-scale simulations.

  14. Characterization of CmaA, an Adenylation-Thiolation Didomain Enzyme Involved in the Biosynthesis of Coronatine

    PubMed Central

    Couch, Robin; O'Connor, Sarah E.; Seidle, Heather; Walsh, Christopher T.; Parry, Ronald

    2004-01-01

    Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-32PPi exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain l-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for l-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4′-phosphopantetheinyl group, reacts with the AMP derivative of l-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and l-[G-3H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the l-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid. PMID:14679222

  15. Characterization of CmaA, an adenylation-thiolation didomain enzyme involved in the biosynthesis of coronatine.

    PubMed

    Couch, Robin; O'Connor, Sarah E; Seidle, Heather; Walsh, Christopher T; Parry, Ronald

    2004-01-01

    Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-(32)PP(i) exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain L-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for L-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4'-phosphopantetheinyl group, reacts with the AMP derivative of L-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and L-[G-(3)H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the L-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid.

  16. Structure of the D-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    SciTech Connect

    Bera, A.K.; Robinson, H.; Atanasova, V.; Gamage, S.; Parsons, J. F.

    2010-06-01

    The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound D-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

  17. Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin

    PubMed Central

    Masin, Jiri; Osickova, Adriana; Sukova, Anna; Fiser, Radovan; Halada, Petr; Bumba, Ladislav; Linhartova, Irena; Osicka, Radim; Sebo, Peter

    2016-01-01

    The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The ‘AC to Hly-linking segment’ thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins. PMID:27581058

  18. Structure of the d-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    SciTech Connect

    Bera, Asim K.; Atanasova, Vesna; Gamage, Swarna; Robinson, Howard; Parsons, James F.

    2010-06-01

    The structure of EhpF from P. agglomerans has been solved alone and in complex with phenazine-1,6-dicarboxylate. Apo EhpF was solved and refined in two different space groups at 1.95 and 2.3 Å resolution and the EhpF–phenazine-1,6-dicarboxylate complex structure was determined at 2.8 Å resolution. The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound d-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

  19. The Adenylate-Forming Enzymes AfeA and TmpB Are Involved in Aspergillus nidulans Self-Communication during Asexual Development

    PubMed Central

    Soid-Raggi, Gabriela; Sánchez, Olivia; Ramos-Balderas, Jose L.; Aguirre, Jesús

    2016-01-01

    Aspergillus nidulans asexual sporulation (conidiation) is triggered by different environmental signals and involves the differentiation of specialized structures called conidiophores. The elimination of genes flbA-E, fluG, and tmpA results in a fluffy phenotype characterized by delayed conidiophore development and decreased expression of the conidiation essential gene brlA. While flbA-E encode regulatory proteins, fluG and tmpA encode enzymes involved in the biosynthesis of independent signals needed for normal conidiation. Here we identify afeA and tmpB as new genes encoding members the adenylate-forming enzyme superfamily, whose inactivation cause different fluffy phenotypes and decreased conidiation and brlA expression. AfeA is most similar to unknown function coumarate ligase-like (4CL-Lk) enzymes and consistent with this, a K544N active site modification eliminates AfeA function. TmpB, identified previously as a larger homolog of the oxidoreductase TmpA, contains a NRPS-type adenylation domain. A high degree of synteny in the afeA-tmpA and tmpB regions in the Aspergilli suggests that these genes are part of conserved gene clusters. afeA, tmpA, and tmpB double and triple mutant analysis as well as afeA overexpression experiments indicate that TmpA and AfeA act in the same conidiation pathway, with TmpB acting in a different pathway. Fluorescent protein tagging shows that functional versions of AfeA are localized in lipid bodies and the plasma membrane, while TmpA and TmpB are localized at the plasma membrane. We propose that AfeA participates in the biosynthesis of an acylated compound, either a p-cuomaryl type or a fatty acid compound, which might be oxidized by TmpA and/or TmpB, while TmpB adenylation domain would be involved in the activation of a hydrophobic amino acid, which in turn would be oxidized by the TmpB oxidoreductase domain. Both, AfeA-TmpA and TmpB signals are involved in self-communication and reproduction in A. nidulans. PMID:27047469

  20. The Adenylate-Forming Enzymes AfeA and TmpB Are Involved in Aspergillus nidulans Self-Communication during Asexual Development.

    PubMed

    Soid-Raggi, Gabriela; Sánchez, Olivia; Ramos-Balderas, Jose L; Aguirre, Jesús

    2016-01-01

    Aspergillus nidulans asexual sporulation (conidiation) is triggered by different environmental signals and involves the differentiation of specialized structures called conidiophores. The elimination of genes flbA-E, fluG, and tmpA results in a fluffy phenotype characterized by delayed conidiophore development and decreased expression of the conidiation essential gene brlA. While flbA-E encode regulatory proteins, fluG and tmpA encode enzymes involved in the biosynthesis of independent signals needed for normal conidiation. Here we identify afeA and tmpB as new genes encoding members the adenylate-forming enzyme superfamily, whose inactivation cause different fluffy phenotypes and decreased conidiation and brlA expression. AfeA is most similar to unknown function coumarate ligase-like (4CL-Lk) enzymes and consistent with this, a K544N active site modification eliminates AfeA function. TmpB, identified previously as a larger homolog of the oxidoreductase TmpA, contains a NRPS-type adenylation domain. A high degree of synteny in the afeA-tmpA and tmpB regions in the Aspergilli suggests that these genes are part of conserved gene clusters. afeA, tmpA, and tmpB double and triple mutant analysis as well as afeA overexpression experiments indicate that TmpA and AfeA act in the same conidiation pathway, with TmpB acting in a different pathway. Fluorescent protein tagging shows that functional versions of AfeA are localized in lipid bodies and the plasma membrane, while TmpA and TmpB are localized at the plasma membrane. We propose that AfeA participates in the biosynthesis of an acylated compound, either a p-cuomaryl type or a fatty acid compound, which might be oxidized by TmpA and/or TmpB, while TmpB adenylation domain would be involved in the activation of a hydrophobic amino acid, which in turn would be oxidized by the TmpB oxidoreductase domain. Both, AfeA-TmpA and TmpB signals are involved in self-communication and reproduction in A. nidulans.

  1. NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme.

    PubMed

    Fry, D C; Kuby, S A; Mildvan, A S

    1985-08-13

    Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual

  2. Calmodulin independence of human duodenal adenylate cyclase.

    PubMed Central

    Smith, J A; Griffin, M; Mireylees, S E; Long, R G

    1991-01-01

    The calmodulin and calcium dependence of human adenylate cyclase from the second part of the duodenum was assessed in washed particulate preparations of biopsy specimens by investigating (a) the concentration dependent effects of free [Ca2+] on enzyme activity, (b) the effects of exogenous calmodulin on enzyme activity in ethylene glycol bis (b-aminoethyl ether)N,N'-tetra-acetic acid (EGTA) washed particulate preparations, and (c) the effects of calmodulin antagonists on enzyme activity. Both basal (IC50 = 193.75 (57.5) nmol/l (mean (SEM)) and NaF stimulated (IC50 = 188.0 (44.0) nmol/l) adenylate cyclase activity was strongly inhibited by free [Ca2+] greater than 90 nmol/l. Free [Ca2+] less than 90 nmol/l had no effect on adenylate cyclase activity. NaF stimulated adenylate cyclase activity was inhibited by 50% at 2.5 mmol/l EGTA. This inhibition could not be reversed by free Ca2+. The addition of exogenous calmodulin to EGTA (5 mmol/l) washed particulate preparations failed to stimulate adenylate cyclase activity. Trifluoperazine and N-(8-aminohexyl)-5-IODO-1-naphthalene-sulphonamide (IODO 8) did not significantly inhibit basal and NaF stimulated adenylate cyclase activity when measured at concentrations of up to 100 mumol/l. These results suggest that human duodenal adenylate cyclase activity is calmodulin independent but is affected by changes in free [Ca2+]. PMID:1752461

  3. Realtime (31)P NMR Investigation on the Catalytic Behavior of the Enzyme Adenylate kinase in the Matrix of a Switchable Ionic Liquid.

    PubMed

    Rogne, Per; Sparrman, Tobias; Anugwom, Ikenna; Mikkola, Jyri-Pekka; Wolf-Watz, Magnus

    2015-11-01

    The integration of highly efficient enzymatic catalysis with the solvation properties of ionic liquids for an environmentally friendly and efficient use of raw materials such as wood requires fundamental knowledge about the influence of relevant ionic liquids on enzymes. Switchable ionic liquids (SIL) are promising candidates for implementation of enzymatic treatments of raw materials. One industrially interesting SIL is constituted by monoethanol amine (MEA) and 1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU) formed with sulfur dioxide (SO2) as the coupling media (DBU-SO2-MEASIL). It has the ability to solubilize the matrix of lignocellulosic biomass while leaving the cellulose backbone intact. Using a novel (31)P NMR-based real-time assay we show that this SIL is compatible with enzymatic catalysis because a model enzyme, adenylate kinase, retains its activity in up to at least 25 wt % of DBU-SO2-MEASIL. Thus this SIL appears suitable for, for example, enzymatic degradation of hemicellulose.

  4. Regulation of brain adenylate cyclase by calmodulin

    SciTech Connect

    Harrison, J.K.

    1988-01-01

    This thesis examined the interaction between the Ca{sup 2+}-binding protein, calmodulin (CaM), and the cAMP synthesizing enzyme, adenylate cyclase. The regulation of guanyl nucleotide-dependent adenylate cyclase by CaM was examined in a particulate fraction from bovine striatum. CaM stimulated basal adenylate cyclase activity and enhanced the stimulation of the enzyme by GTP and dopamine (DA). The potentiation of GTP- and DA-stimulated adenylate cyclase activities by CaM was more sensitive to the concentration of CaM than was the stimulation of basal activity. A photoreactive CaM derivative was developed in order to probe the interactions between CaM and the adenylate cyclase components of bovine brain. Iodo-({sup 125}I)-CaM-diazopyruvamide ({sup 125}I-CAM-DAP) behaved like native CaM with respect to Ca{sup 2+}-enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and Ca{sup 2+}-dependent stimulation of adenylate cyclase. {sup 125}I-CaM-DAP cross-linked to CaM-binding proteins in a Ca{sup 2+}-dependent, concentration-dependent, and CaM-specific manner. Photolysis of {sup 125}I-CaM-DAP and forskolin-agarose purified CaM-sensitive adenylate cyclase produced an adduct with a molecular weight of 140,000.

  5. Genome scale prediction of substrate specificity for acyl adenylate superfamily of enzymes based on active site residue profiles.

    PubMed

    Khurana, Pankaj; Gokhale, Rajesh S; Mohanty, Debasisa

    2010-01-27

    Enzymes belonging to acyl:CoA synthetase (ACS) superfamily activate wide variety of substrates and play major role in increasing the structural and functional diversity of various secondary metabolites in microbes and plants. However, due to the large sequence divergence within the superfamily, it is difficult to predict their substrate preference by annotation transfer from the closest homolog. Therefore, a large number of ACS sequences present in public databases lack any functional annotation at the level of substrate specificity. Recently, several examples have been reported where the enzymes showing high sequence similarity to luciferases or coumarate:CoA ligases have been surprisingly found to activate fatty acyl substrates in experimental studies. In this work, we have investigated the relationship between the substrate specificity of ACS and their sequence/structural features, and developed a novel computational protocol for in silico assignment of substrate preference. We have used a knowledge-based approach which involves compilation of substrate specificity information for various experimentally characterized ACS and derivation of profile HMMs for each subfamily. These HMM profiles can accurately differentiate probable cognate substrates from non-cognate possibilities with high specificity (Sp) and sensitivity (Sn) (Sn = 0.91-1.0, Sp = 0.96-1.0) values. Using homologous crystal structures, we identified a limited number of contact residues crucial for substrate recognition i.e. specificity determining residues (SDRs). Patterns of SDRs from different subfamilies have been used to derive predictive rules for correlating them to substrate preference. The power of the SDR approach has been demonstrated by correct prediction of substrates for enzymes which show apparently anomalous substrate preference. Furthermore, molecular modeling of the substrates in the active site has been carried out to understand the structural basis of substrate selection. A web

  6. Dynamics in Thermotoga neapolitana adenylate kinase: 15N relaxation and hydrogen-deuterium exchange studies of a hyperthermophilic enzyme highly active at 30 degrees C.

    PubMed

    Krishnamurthy, Harini; Munro, Kim; Yan, Honggao; Vieille, Claire

    2009-03-31

    Backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A) were investigated at 30 degrees C using (15)N NMR relaxation measurements and NMR monitored hydrogen-deuterium exchange. With kinetic parameters identical to those of Escherichia coli AK (ECAK) at 30 degrees C, TNAK is a unique hyperthermophilic enzyme. These catalytic properties make TNAK an interesting and novel model to study the interplay between protein rigidity, stability, and activity. Comparison of fast time scale dynamics (picosecond to nanosecond) in the open and closed states of TNAK and ECAK at 30 degrees C reveals a uniformly higher rigidity across all domains of TNAK. Within this framework of a rigid TNAK structure, several residues located in the AMP-binding domain and in the core-lid hinge regions display high picosecond to nanosecond time scale flexibility. Together with the recent comparison of ECAK dynamics with those of hyperthermophilic Aquifex aeolicus AK (AAAK), our results provide strong evidence for the role of picosecond to nanosecond time scale fluctuations in both stability and activity. In the slow time scales, TNAK's increased rigidity is not uniform but localized in the AMP-binding and lid domains. The core domain amides of ECAK and TNAK in the open and closed states show comparable protection against exchange. Significantly, the hinges framing the lid domain show similar exchange data in ECAK and TNAK open and closed forms. Our NMR relaxation and hydrogen-deuterium exchange studies therefore suggest that TNAK maintains high activity at 30 degrees C by localizing flexibility to the hinge regions that are key to facilitating conformational changes.

  7. Biochemical and Structural Characterization of Bisubstrate Inhibitors of BasE, the Self-standing Non-Ribosomal Peptide Synthetase Adenylate-Forming Enzyme of Acinetobactin Synthesis†,‡

    PubMed Central

    Drake, Eric J.; Duckworth, Benjamin P.; Neres, João; Aldrich, Courtney C.; Gulick, Andrew M.

    2010-01-01

    The human pathogen Acinetobacter baumannii produces a siderophore called acinetobactin that is derived from one molecule each of threonine, histidine, and 2,3-dihydroxybenzoic acid (DHB). The activity of several non-ribosomal peptide synthetase (NRPS) enzymes is used to combine the building blocks into the final molecule. The acinetobactin synthesis pathway initiates with a self-standing adenylation enzyme, BasE, that activates the DHB molecule and covalently transfers it to the pantetheine cofactor of an aryl-carrier protein of BasF, a strategy that is shared with many siderophore-producing NRPS clusters. In this reaction, DHB reacts with ATP to form the aryl adenylate and pyrophosphate. In a second partial reaction, the DHB is transferred to the carrier protein. Inhibitors of BasE and related enzymes have been identified that prevent growth of bacteria on iron-limiting media. Recently, a new inhibitor of BasE has been identified via high-throughput screening using a fluorescence polarization displacement assay. We present here biochemical and structural studies to examine the binding mode of this inhibitor. The kinetics of the wild-type BasE enzyme is shown and inhibition studies demonstrate that the new compound exhibits competitive inhibition against both ATP and 2,3-dihydroxybenzoate. Structural examination of BasE bound to this inhibitor illustrates a novel binding mode in which the phenyl moiety partially fills the enzyme pantetheine binding tunnel. Structures of rationally designed bisubstrate inhibitors are also presented. PMID:20853905

  8. 33 CFR 165.T01-0048 - Regulated Navigation Area; MBTA Saugus River Railroad Drawbridge rehabilitation project, Saugus...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... WATERWAYS SAFETY REGULATED NAVIGATION AREAS AND LIMITED ACCESS AREAS Specific Regulated Navigation Areas and... a Regulated Navigation Area (RNA): All navigable waters, surface to bottom, on the Saugus River... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Regulated Navigation Area; MBTA...

  9. Studies on the adenylate kinase isozymes from the serum and erythrocyte of normal and Duchenne dystrophic patients. Isolation, physicochemical properties, and several comparisons with the Duchenne dystrophic aberrant enzyme.

    PubMed

    Hamada, M; Sumida, M; Kurokawa, Y; Sunayashiki-Kusuzaki, K; Okuda, H; Watanabe, T; Kuby, S A

    1985-09-25

    Two species of adenylate kinase isozymes (ATP:AMP phosphotransferase, EC 2.7.4.3) from human Duchenne dystrophic serum were separated by Blue Sepharose CL-6B affinity column chromatography. One of these species was the "aberrant" adenylate kinase isozyme, found specifically in the Duchenne type of this disease (Hamada, M., Okuda, H., Oka, K., Watanabe, T., Ueda, K., Nojima, M., Kuby, S.A., Manship, M., Tyler, F., and Ziter, F. (1981) Biochim. Biophys. Acta 660, 227-237). The separated aberrant form possessed a molecular size of 98,000 (+/- 1,500), whereas the normal serum species of the enzyme was 87,000 (+/- 1,600) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by gel filtration, and by sedimentation equilibrium. The sedimentation coefficient of each species was found to be 5.8 S for the aberrant form and 5.6 S for the normal form, respectively. The subunit size (Mr = 24,700) of the aberrant enzyme in 8 M urea proved to be very similar to that of the normal human liver enzyme (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S.A. (1982) J. Biol. Chem. 257, 13120-13128), and the normal species subunit (Mr = 21,700) was found to be very similar to that of the normal human muscle enzyme (Kuby, S.A., Fleming, G., Frischat, A., Cress, M.C., and Hamada, M. (1983) J. Biol. Chem. 258, 1901-1907). Both species were tetrameric enzymes in the serum. The amino acid composition for the normal species was similar to that for the muscle-type enzyme, and that for the aberrant species was similar to the liver enzyme, but with some notable exceptions in both cases. Thus, the normal species had no tryptophan and two half-cystine residues/subunit; whereas, there was 1 tryptophan and 4 half-cystine residues/subunit of the aberrant molecule. The amino acid composition of both serum isozymes when compared to their respective muscle or liver-type enzyme differed mainly in the content of Glu, Asp, His, Leu, Ile, Gly. Kinetic properties of the two forms

  10. Identification of the Fluvirucin B2 (Sch 38518) Biosynthetic Gene Cluster from Actinomadura fulva subsp. indica ATCC 53714: substrate Specificity of the β-Amino Acid Selective Adenylating Enzyme FlvN.

    PubMed

    Miyanaga, Akimasa; Hayakawa, Yuki; Numakura, Mario; Hashimoto, Junko; Teruya, Kuniko; Hirano, Takashi; Shin-Ya, Kazuo; Kudo, Fumitaka; Eguchi, Tadashi

    2016-05-01

    Fluvirucins are 14-membered macrolactam polyketides that show antifungal and antivirus activities. Fluvirucins have the β-alanine starter unit at their polyketide skeletons. To understand the construction mechanism of the β-alanine moiety in fluvirucin biosyntheses, we have identified the biosynthetic cluster of fluvirucin B2 produced from Actinomadura fulva subsp. indica ATCC 53714. The identified gene cluster contains three polyketide synthases, four characteristic β-amino acid-carrying enzymes, one decarboxylase, and one amidohydrolase. We next investigated the activity of the adenylation enzyme FlvN, which is a key enzyme for the selective incorporation of a β-amino acid substrate. FlvN showed strong preference for l-aspartate over other amino acids such as β-alanine. Based on these results, we propose a biosynthetic pathway for fluvirucin B2.

  11. Plant adenylate cyclases.

    PubMed

    Lomovatskaya, Lidiya A; Romanenko, Anatoliy S; Filinova, Nadejda V

    2008-01-01

    Adenylate cyclase (AC) (ATP diphosphate-lyase cyclizing; EC 4.6.1.1) is a key component of the adenylate cyclase signaling system and catalyzes the generation of cyclic adenosine monophosphate (cAMP) from ATP. This review summarizes data from the literature and the authors' laboratory on the investigation of plant transmembrane (tmAC) and soluble (sAC) adenylate cyclases, in comparison with some key characteristics of adenylate cyclases of animal cells. Plant sAC has been demonstrated to exhibit similarities with animal sAC with respect to certain characteristics. External factors, such as far-red and red light, temperature, exogenous phytohormones, as well as specific triggering compounds of fungal and bacterial origin exert a significant influence on the activity of plant tmAC and sAC.

  12. Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa).

    PubMed Central

    Carricarte, V C; Bianchini, G M; Muschietti, J P; Téllez-Iñón, M T; Perticari, A; Torres, N; Flawiá, M M

    1988-01-01

    An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase. PMID:3128270

  13. Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay.

    PubMed

    Duckworth, Benjamin P; Wilson, Daniel J; Aldrich, Courtney C

    2016-01-01

    Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.

  14. Adenosine tetraphosphoadenosine drives a continuous ATP-release assay for aminoacyl-tRNA synthetases and other adenylate-forming enzymes.

    PubMed

    Lloyd, Adrian J; Potter, Nicola J; Fishwick, Colin W G; Roper, David I; Dowson, Christopher G

    2013-10-18

    Aminoacyl-tRNA synthetases are essential for the correct linkage of amino acids to cognate tRNAs to maintain the fidelity of protein synthesis. Tractable, continuous assays are valuable for characterizing the functions of synthetases and for their exploitation as drug targets. We have exploited the unexplored ability of these enzymes to consume adenosine tetraphosphoadenosine (diadenosine 5',5‴ P(1) P(4) tetraphosphate; Ap4A) and produce ATP to develop such an assay. We have used this assay to probe the stereoselectivity of isoleucyl-tRNA(Ile) and Valyl-tRNA(Val) synthetases and the impact of tRNA on editing by isoleucyl-tRNA(Ile) synthetase (IleRS) and to identify analogues of intermediates of these enzymes that might allow targeting of multiple synthetases. We further report the utility of Ap4A-based assays for identification of synthetase inhibitors with nanomolar to millimolar affinities. Finally, we demonstrate the broad application of Ap4A utilization with a continuous Ap4A-driven RNA ligase assay.

  15. Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase

    PubMed Central

    2016-01-01

    AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target. PMID:27088128

  16. An aberrant adenylate kinase isoenzyme from the serum of patients with Duchenne muscular dystrophy.

    PubMed

    Hamada, M; Okuda, H; Oka, K; Watanabe, T; Ueda, K; Nojima, M; Kuby, S A; Manship, M; Tyler, F H; Ziter, F A

    1981-08-13

    The sera from patients with human Duchenne (X-linked) progressive muscular dystrophy contain elevated adenylate kinase (ATP: AMP phosphotransferase, EC 2.7.4.3) activities, in addition to their characteristically high creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) activities. By agarose gel electrophoresis of human Duchenne dystrophic serum, the presence of an apparently normal human serum adenylate kinase together with a variant species of adenylate kinase was detected. The latter enzyme species appeared, in its mobility, to be similar to that of the normal human liver-type adenylate kinase. The presence of this aberrant liver-type adenylate kinase could also be demonstrated by characteristic (for the liver type) inhibition patterns with P1,P5-di-(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate. On the other hand, by inhibition titrations with an anti-muscle-type adenylate kinase, hemolysates from the erythrocytes of several Duchenne and Becker's dystrophics were found to contain approx. 96% muscle-type adenylate kinase and their serum approx. 97% muscle-type adenylate kinase. These same patients contained approx. 89% M-M type creatine kinase in their serum (by inhibition against anti-human muscle-type creatine kinase) indicative of the presence also of M-B plus B-B type active isoenzymes. All of these data can best be explained by the presence of a variant or mutant adenylate kinase isoenzyme in the dystrophic serum. This isoenzyme appears to resemble the liver type in its inhibition patterns with P1,P5-di(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate, and in its heat stability (compare also the agarose gel electrophoresis pattern); but structurally, it is a muscle type, or derived from a muscle type, as shown immunologically by inhibition reactions with anti-muscle-type adenylate kinase. Whether this is a fetal-type isoenzyme of adenylate kinase will require further

  17. Glucose Inhibition of Adenylate Cyclase in Intact Cells of Escherichia coli B

    PubMed Central

    Peterkofsky, Alan; Gazdar, Celia

    1974-01-01

    Previous studies in E. coli B have demonstrated an inverse correlation between the presence of glucose in the medium and the accumulation of cyclic AMP in the medium. This observation could not be explained by the action of glucose as a repressor of adenylate cyclase (EC 4.6.1.1) synthesis, as a stabilizer of cyclic AMP phosphodiesterase (EC 3.1.4.17) activity, or as a direct inhibitor of adenylate cyclase activity in cell-free preparations. The recent development of an in vivo assay for adenylate cyclase has provided a basis for further exploring the inhibitory action of glucose in intact cells. With this assay it has been possible to show that, while glucose does not affect adenylate cyclase in vitro, it rapidly inhibits the enzyme activity in intact cells. Extensive metabolism of glucose is not required, since α-methylglucoside also inhibits adenylate cyclase in vivo. When cells are grown on glucose as carbon source, some sugars (mannose, glucosamine) substitute for glucose as adenylate cyclase inhibitors while others (e.g., fructose) do not. Dose-response studies indicate that low concentrations of glucose lead to essentially complete inhibition of adenylate cyclase activity while only moderately decreasing intracellular cyclic AMP concentrations. The evidence presented suggests that the decreased cellular cyclic AMP levels resulting from glucose addition can be accounted for by inhibition of adenylate cyclase without any significant effect on cyclic AMP phosphodiesterase or the transport of cyclic AMP from the cells to the medium. PMID:4366761

  18. Adenylate cyclases involvement in pathogenicity, a minireview.

    PubMed

    Costache, Adriana; Bucurenci, Nadia; Onu, Adrian

    2013-01-01

    Cyclic AMP (cAMP), one of the most important secondary messengers, is produced by adenylate cyclase (AC) from adenosine triphosphate (ATP). AC is a widespread enzyme, being present both in prokaryotes and eukaryotes. Although they have the same enzymatic activity (ATP cyclization), the structure of these proteins varies, depending on their function and the producing organism. Some pathogenic bacteria utilize these enzymes as toxins which interact with calmodulin (or another eukaryote activator), causing intense cAMP synthesis and disruption of infected cell functions. In contrast, other pathogenic bacteria benefit of augmentation of AC activity for their own function. Based on sequence analysis ofAC catalytic domain from two pathogenic bacteria (Bacillus anthracis and Bordetellapertussis) with known three-dimensional structures, a possible secondary structure for 1-255 amino acid fragment from Pseudomonas aeruginosa AC (with 80TKGFSVKGKSS90 as the ATP binding site) is proposed.

  19. Food restriction modulates. beta. -adrenergic-sensitive adenylate cyclase in rat liver during aging

    SciTech Connect

    Katz, M.S. Audie L. Murphy Memorial Veterans Hospital, San Antonio, TX )

    1988-01-01

    Adenylate cyclase activities were studied in rat liver during postmaturational aging of male Fischer 344 rats fed ad libitum or restricted to 60% of the ad libitum intake. Catecholamine-stimulated adenylate cyclase activity increased by 200-300% between 6 and 24-27 mo of age in ad libitum-fed rats, whereas in food-restricted rats catecholamine response increased by only 58-84% between 6 and 30 mo. In ad libitum-fed rats, glucagon-stimulated enzyme activity also increased by 40% between 6 and 12 mo and in restricted rats a similar age-related increase was delayed until 18 mo. {beta}-Adrenergic receptor density increased by 50% between 6 and 24 mo in livers from ad libitum-fed but not food-restricted rats and showed a highly significant correlation with maximal isoproterenol-stimulated adenylate cyclase activity over the postmaturational life span. Age-related increases in unstimulated (basal) adenylate cyclase activity and nonreceptor-mediated enzyme activation were retarded by food restriction. The results demonstrate that food restriction diminishes a marked age-related increase in {beta}-adrenergic-sensitive adenylate cyclase activity of rat liver. Alterations of adrenergic-responsive adenylate cyclase with age and the modulatory effects of food restriction appear to be mediated by changes in both receptor and nonreceptor components of adenylate cyclase.

  20. Forskolin activation of serotonin-stimulated adenylate cyclase in the liver fluke Fasciola hepatica.

    PubMed

    McNall, S J; Mansour, T E

    1985-05-15

    Properties of forskolin activation of adenylate cyclase in the liver fluke Fasciola hepatica are described. Forskolin stimulated adenylate cyclase activity in cell-free fluke particles to levels more than 30-fold above the basal rate. This activation was not dependent on guanine nucleotides and, upon washing of the particles, was rapidly reversed. Forskolin potentiated the activation of adenylate cyclase by serotonin (5-HT) and lysergic acid diethylamide (LSD), resulting in both an increase in the maximal level of enzyme activity and a decrease in the apparent activation constant (KA). The 5-HT antagonist 2-bromo-LSD did not inhibit enzyme activation by forskolin. Furthermore, forskolin had no effect on specific [3H]LSD binding to fluke particles. Activation of adenylate cyclase by sodium fluoride or guanine nucleotides was modified in a complex manner by forskolin with both stimulatory and inhibitory effects present. The results suggest that forskolin does not interact directly with the 5-HT receptor coupled to adenylate cyclase. Instead, it appears that forskolin effects are, at least in part, due to its ability to alter the interaction between the regulatory and catalytic components of adenylate cyclase. Incubation of intact flukes with forskolin increased their cAMP levels 2- to 3-fold. The concentration dependence of this response was similar to that for forskolin activation of adenylate cyclase in fluke particles, with 300 microM forskolin giving the maximum response. Forskolin and other agents that increased fluke cAMP levels also stimulated fluke motility.

  1. Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand

    PubMed Central

    Patel, Maha P.; Baum, Dana A.; Silverman, Scott K.

    2008-01-01

    DNA with a 5′-adenylpyrophosphoryl cap (5′-adenylated DNA; AppDNA) is an activated form of DNA that is the biochemical intermediate of the reactions catalyzed by DNA ligase, RNA ligase, polynucleotide kinase, and other nucleic acid modifying enzymes. 5′-Adenylated DNA is also useful for in vitro selection experiments. Efficient preparation of 5′-adenylated DNA is therefore desirable for several biochemical applications. Here we have developed a DNA adenylation procedure that uses T4 DNA ligase and is more reliable than a previously reported approach that used the 5′-phosphorylated donor DNA substrate to be adenylated, a DNA template, and ATP but no acceptor strand. Our improved DNA adenylation procedure uses the above components as well as an acceptor strand that has a strategically chosen C-T acceptor-template mismatch directly adjacent to the adenylation site. This mismatch permits adenylation of the donor DNA substrate but largely suppresses subsequent ligation of the donor with the acceptor, as assayed on nine different DNA substrates that collectively have all four DNA nucleotides represented at each of the first two positions. The new DNA adenylation procedure is successful using either laboratory-prepared or commercial T4 DNA ligase and works well on the preparative (2 nmol) scale for all nine of the test DNA substrates. PMID:18022669

  2. Independent sensitization of β-adrenoceptors and adenylate cyclase in acute myocardial ischaemia

    PubMed Central

    Strasser, R. H.; Marquetant, R.; Kübler, W.

    1990-01-01

    1 Acute myocardial ischaemia provokes sensitization of the adenylate cyclase system. This sensitization could be differentiated in a receptor-linked and an enzyme-linked sensitization. The increase in the number of β-adrenoceptors in the plasma membranes was observed already after 15 min of global ischaemia (50 ± 2 to 67 ± 6 fmol mg-1 protein) and persisted after 50 min of ischaemia. The maximally isoprenaline-stimulated adenylate cyclase activity rose from 66 ± 7 to 100 ± 10 pmol cAMP min-1 mg-1 protein after 15 min of global ischaemia indicating the receptor-mediated sensitization of the β-adrenergic system. However, after 50 min of ischaemia the isoprenaline-stimulated adenylate cyclase was reduced by about 50% despite the continuous increase of β-adrenoceptors in the plasma membranes. 2 Additionally direct stimulation of the adenylate cyclase by forskolin revealed an increased enzyme activity after 15 min of global ischaemia (300 ± 20 vs 378 ± 25 pmol cAMP min-1 mg-1). Prolonged periods of ischaemia, however, caused a decline of the total adenylate cyclase activity (232 ± 24 pmol cAMP min-1 mg-1 protein). This demonstrates an enzyme-specific sensitization of the adenylate cyclase, which in contrast to the rise in β-adrenoceptors is only transient. This enzyme-specific sensitization or the late inactivation of the enzyme occur independently of receptor activation and cannot be prevented by β-adrenoceptor blockade (10-6 M alprenolol) prior to the ischaemic insult. For the first time it could be demonstrated that the enzyme-linked sensitization is carried by the adenylate cyclase even after partial purification of the enzyme including solubilization and wheatgerm affinity chromatography. These data may suggest an ischaemia-induced covalent modification of the adenylate cyclase. The enzyme-linked sensitization and the late inactivation of the enzyme do not occur after cyanide perfusion demonstrating that energy depletion is not solely responsible for

  3. Characterization of the purine-reactive site of the rat testis cytosolic adenylate cyclase.

    PubMed

    Onoda, J M; Braun, T; Wrenn, S M

    1987-06-15

    Naturally soluble rat germ cell adenylate cyclase was inhibited by adenosine and the adenosine analogs, 9-beta-D-arabinofuranosyl adenine (AFA) and 2',5'-dideoxyadenosine (DDA), all of which inhibited hormone-sensitive adenylate cyclases at the "P" site. The IC50 values for adenosine and DDA were approximately 0.1 and for AFA, 4.0 mM. The onset of adenosine inhibition was very rapid whether adenosine was added to the enzyme reactant mixture at time zero concomitantly with the addition of substrate or after the enzyme had been activated by the addition of substrate. The adenosine analogs, N6-methyladenosine (MeA) and N6-phenylisopropyl adenosine (PIA), which interact with plasma membrane receptors ("R" receptors) for hormone-sensitive adenylate cyclase, had little effect on the activity of the cytosolic adenylate cyclase. Additionally, aminophylline, which has been shown to competitively antagonize adenosine interactions with the plasma membrane "R" receptors but not "P" site interactions, had no effect upon substrate activation of the soluble enzyme and did not prevent adenosine from inhibiting the activity of the enzyme. These data provide evidence for an adenosine regulatory site on the cytosolic enzyme which resembles the "P" site described for membrane bound-adenylate cyclase.

  4. Dopaminergic modulation of adenylate cyclase stimulation by vasoactive intestinal peptide in anterior pituitary.

    PubMed Central

    Onali, P; Schwartz, J P; Costa, E

    1981-01-01

    The activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by vasoactive intestinal peptide (VIP) was used as a model to investigate the molecular mechanisms triggered by the occupancy of dopamine recognition sites in rat anterior pituitary. Dopamine failed to change the basal enzyme activity, but it inhibited the stimulation of adenylate cyclase elicited by VIP. Apomorphine, 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene, and 2-bromo-alpha-ergocryptine mimicked the effect of dopamine, whereas (-)-sulpiride and and classical neuroleptics antagonized it. Dopamine failed to modulate the activation of pituitary adenylate cyclase by prostaglandin E1, which does not increase prolactin secretion. From these results we infer that stimulation of D-2 (dopamine) receptors may affect pituitary secretion by inhibiting the activation of anterior pituitary adenylate cyclase by VIP or other secretagogues. PMID:6171819

  5. Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

    PubMed Central

    Kalb, Daniel; Lackner, Gerald

    2014-01-01

    A key step in fungal l-lysine biosynthesis is catalyzed by adenylate-forming l-α-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional in vitro characterization of four reductases which were heterologously produced in Escherichia coli. The Ceriporiopsis subvermispora serine reductase Nps1 features a terminal ferredoxin-NADP+ reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by Ceriporiopsis subvermispora Nps3 as the first biochemically characterized l-α-aminoadipic acid reductase of basidiomycete origin. Aspergillus flavus l-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes. PMID:25085485

  6. Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain

    PubMed Central

    González-Bullón, David; Uribe, Kepa B.; Martín, César

    2017-01-01

    Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacterium Bordetella pertussis. Secreted as soluble protein, it targets myeloid cells expressing the CD11b/CD18 integrin and on delivery of its N-terminal adenylate cyclase catalytic domain (AC domain) into the cytosol, generates uncontrolled toxic levels of cAMP that ablates bactericidal capacities of phagocytes. Our study deciphers the fundamentals of the heretofore poorly understood molecular mechanism by which the ACT enzyme domain directly crosses the host cell membrane. By combining molecular biology, biochemistry, and biophysics techniques, we discover that ACT has intrinsic phospholipase A (PLA) activity, and that such activity determines AC translocation. Moreover, we show that elimination of the ACT–PLA activity abrogates ACT toxicity in macrophages, particularly at toxin concentrations close to biological reality of bacterial infection. Our data support a molecular mechanism in which in situ generation of nonlamellar lysophospholipids by ACT–PLA activity into the cell membrane would form, likely in combination with membrane-interacting ACT segments, a proteolipidic toroidal pore through which AC domain transfer could directly take place. Regulation of ACT–PLA activity thus emerges as novel target for therapeutic control of the disease. PMID:28760979

  7. The energy landscape of adenylate kinase during catalysis

    PubMed Central

    Kerns, S. Jordan; Agafonov, Roman V.; Cho, Young-Jin; Pontiggia, Francesco; Otten, Renee; Pachov, Dimitar V.; Kutter, Steffen; Phung, Lien A.; Murphy, Padraig N.; Thai, Vu; Alber, Tom; Hagan, Michael F.; Kern, Dorothee

    2014-01-01

    Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, MD simulations, and crystallography of active complexes. We find that the Mg2+ cofactor activates two distinct molecular events, phosphoryl transfer (>105-fold) and lid-opening (103-fold). In contrast, mutation of an essential active-site arginine decelerates phosphoryl transfer 103-fold without substantially affecting lid-opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a pre-organized active site. PMID:25580578

  8. [Isoenzymatic forms and distribution of adenylate kinase and creatine kinase in the bovine adrenal medulla].

    PubMed

    Miras-Portugal, M T; Orera, A; Millaruelo, A

    1982-01-01

    Adenylate kinase, creatine kinase as well as their substrate and product levels have been investigated in the adrenal medullary tissue. The concentration of adenine nucleotides and creatine + creatine phosphate are 12.6 +/- 0.4 and 6.9 +/- 0.4 mumol/g wet weight respectively. Adenylate kinase is mainly in the cytosol; only 4% was found in mitochondria. The cytosol enzyme presents a Km for AMP of 5 X 10(-4) M and a Ki for diadenosine pentaphosphate of 0.6 X 10(-6) M. In gel electrophoresis, only one band of adenylate kinase activity can be seen, and its mobility is different from that of the brain enzyme. Creatine kinase from adrenal medulla is mainly found in cytosol; only 3-4% was associated with mitochondria. The cytosolic enzyme is mainly the BB isozyme form.

  9. The invasive adenylate cyclase of Bordetella pertussis. Properties and penetration kinetics.

    PubMed Central

    Friedman, E; Farfel, Z; Hanski, E

    1987-01-01

    Bordetella pertussis, the causative organism of whooping cough, produces a calmodulin-sensitive adenylate cyclase. Confer & Eaton [(1982) Science 217, 948-950] have shown that an extract from B. pertussis increases intracellular cyclic AMP levels in neutrophils and suggested that this increase is caused by the bacterial adenylate cyclase which penetrates these cells. We demonstrate in the present study that adenylate cyclase activity in lysates from lymphocytes exposed to a partially purified preparation of the bacterial enzyme has properties completely different from those of the intrinsic membrane-bound enzyme. Adenylate cyclase activity in lysates from lymphocytes exposed to the invasive enzyme is insensitive to N-ethylmaleimide, readily inactivated by acetic anhydride and relatively stable to SDS. Similar properties are exhibited by the bacterial enzyme itself. By contrast, the intrinsic membrane-bound enzyme activated by forskolin and guanosine 5'-gamma-thiotriphosphate is sensitive to N-ethylmaleimide and SDS and relatively stable to acetic anhydride. This strongly supports the notion that B. pertussis adenylate cyclase penetrates cells. Using the partially purified preparation of the invasive enzyme, we have studied the kinetics of its penetration. The intracellular catalytic activity reaches a steady state within 20 min, irrespective of enzyme or cell concentration. Steady-state levels are maintained for at least 2 h provided that the invasive enzyme is present in the incubation medium. Upon its removal, a rapid decrease (t1/2 approximately equal to 15 min) in the intracellular cyclase level is observed. This decrease reflects intracellular inactivation of the bacterial enzyme and is not caused by the release of the enzyme to the cell medium. PMID:2886119

  10. Developmental changes of beta-adrenergic receptor-linked adenylate cyclase of rat liver

    SciTech Connect

    Katz, M.S.; Boland, S.R.; Schmidt, S.J.

    1985-06-01

    beta-Adrenergic agonist-sensitive adenylate cyclase activity and binding of the beta-adrenergic antagonist(-)-(/sup 125/I)iodopindolol were studied in rat liver during development of male Fischer 344 rats ages 6-60 days. In liver homogenates maximum adenylate cyclase response to beta-adrenergic agonist (10(-5) M isoproterenol or epinephrine) decreased by 73% (P less than 0.01) between 6 and 60 days, with most of the decrease (56%; P less than 0.01) occurring by 20 days. beta-adrenergic receptor density (Bmax) showed a corresponding decrease of 66% (P less than 0.01) by 20 days without subsequent change. Binding characteristics of stereospecificity, pharmacological specificity, saturability with time, and reversibility were unchanged with age. GTP-, fluoride-, forskolin-, and Mn2+-stimulated adenylate cyclase activities also decreased during development, suggesting a decrease of activity of the catalytic component and/or guanine nucleotide regulatory component of adenylate cyclase. These results indicate that the developmental decrease of beta-adrenergic agonist-sensitive adenylate cyclase activity may result from decreased numbers of beta-adrenergic receptors. Developmental alterations of nonreceptor components of the enzyme may also contribute to changes of catecholamine-sensitive adenylate cyclase.

  11. Modification of adenylate cyclase by photoaffinity analogs of forskolin

    SciTech Connect

    Ho, L.T.; Nie, Z.M.; Mende, T.J.; Richardson, S.; Chavan, A.; Kolaczkowska, E.; Watt, D.S.; Haley, B.E.; Ho, R.J. )

    1989-01-01

    Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking (125I)PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by (125I)PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that (a) no other AC-regulatory proteins are known to be of this size, (b) the catalytic unit of bovine brain enzyme is in the same range and (c) this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.

  12. Properties of Adenyl Cyclase from Human Jejunal Mucosa during Naturally Acquired Cholera and Convalescence

    PubMed Central

    Chen, Lincoln C.; Rohde, Jon E.; Sharp, Geoffrey W. G.

    1972-01-01

    The enterotoxin of Vibrio cholerae causes copious fluid production throughout the lenght of the small intestine. As this is thought to be mediated by stimulation of adenyl cyclase, a study has been made of the activity and properties of this enzyme in jejunal biopsy tissue taken from patients during the diarrheal phase of cholera and after recovery. Adenyl cyclase activity during cholera was increased more than twofold relative to the enzyme in convalescence. Under both conditions stimulation by prostaglandin E1 (PGE1) and by fluoride was observed. The responsiveness to PGE1 was not altered in cholera; the total activity of the fluoride-stimulated enzyme was similar, a finding that suggests cholera toxin stimulates pre-existing enzyme in the intestinal cell. The enzymes during cholera and convalescence were similar in all other properties examined. Optimal Mg++ concentration was 10 mM; Mn++ at 5 mM stimulated the enzyme but could not replace Mg++ except in the presence of 10 mM fluoride. Calcium was markedly inhibitory at concentrations greater than 10-4 M. The pH optimum was 7.5 and the Michaelis constant (Km) for ATP concentration approximated 10-4 M. Thus the interaction of cholera toxin with human intestinal adenyl cyclase does not alter the basic properties of the enzyme. When biopsy specimens were maintained intact in oxygenated Ringer's solution at 0°C, no loss of activity was observed at 1½ and 3 hr. In contrast, when the cells were homogenized, rapid loss of activity, with a half-life of 90 min was seen even at 0°C. Consequently for comparative assays of human jejunal adenyl cyclase, strict control of the experimental conditions is required. It was under such conditions that a twofold increase in basal adenyl cyclase activity during cholera was observed. Images PMID:4335441

  13. The flagellar adenylate kinases of Trypanosoma cruzi.

    PubMed

    Camara, María de los Milagros; Bouvier, León A; Miranda, Mariana R; Pereira, Claudio A

    2015-01-01

    Adenylate kinases (ADK) are key enzymes involved in cell energy management. Trypanosomatids present the highest number of variants in a single cell in comparison with the rest of the living organisms. In this work, we characterized two flagellar ADKs from Trypanosoma cruzi, called TcADK1 and TcADK4, which are also located in the cell cytosol. Interestingly, TcADK1 presents a stage-specific expression. This variant was detected in epimastigotes cells, and was completely absent in trypomastigotes and amastigotes, while TcADK4 is present in the major life cycle stages of T. cruzi. Both variants are also regulated, in opposite ways, along the parasite growth curve suggesting that their expression depends on the intra- and extracellular conditions. Both, TcADK1 and TcADK4 present N-terminal extension that could be responsible for their subcellular localization. The presence of ADK variants in the flagellum would be critical for the provision of energy in a process of high ATP consumption such as cell motility. © The Author 2014. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Adenyl cyclase in the human placenta.

    PubMed

    Sato, K; Ryan, K J

    1971-09-21

    This study demonstrated that the human placenta possesses an adenyl cyclase system responsive to catecholamines and sodium flouride (NaF). 2.5 gm human term placentas were homogenized, centrifuged, washed, resuspended, and used as the enzyme system when placed with various agents. Incubations and the determination of adenosine 3', 5' monophosphate (cyclic AMP) formed were performed. Samples stimulated by .0001 M catecholamines (L-epinephrine or L-norepinephrine) or .01 M NaF had higher levels of cyclic AMP than the controls (p. 005 for catecholamine-treated samples and p. 001 for NaF-treated samples). A concentration of .0001 M L-epinephrine or L-norepinephrine appeared to be a maximum effective dose and .0000001 M a minimum. L=epinephrine was 10 times as effective in the stimulation as L-norepinephrine. With .0001 M, 499 and 439 pmoles/10 minutes per 25 mg of tissue was formed, whereas in the control (no added hormones) 256 pmoles/10 minutes were formed. 3.2% ethanol activated the system by a small amount (p.02). Propranolol alone did not appear to have any effect; however, the effect of .0001 M L-epinephrine was reduced by 95% in the presence of .00001 M propranolol. Propranolol had no effect on NaF-stimulated activity.

  15. Microscopical localization on adenylate cyclase: a historical review of methodologies.

    PubMed

    Richards, P A; Richards, P D

    1998-03-15

    The histochemistry technique for localizing adenylate cyclase has been developed over the past two decades. Early efforts were directed at overcoming the criticism of the lead capture technique, the inhibition of the enzyme by fixation, and problems associated with the substrate. The introduction of alternative metal ions, strontium and cerium, offered solutions to the criticism of the lead capture technique. The inhibition of the enzyme by the various fixation methods used has been rarely overcome satisfactorily and the use of non-fixed material during incubation is one of the alternatives that has been suggested. The introduction of adenylate (beta-gamma-methylene) diphosphate as an alternative substrate offers a solution to the problems associated with commercially available adenylyl imidodiphosphate. Although no standard medium or method has been accepted by all researchers, the histochemical technique still has a place in the arsenal of the modern cell biologist. The technique localizes the active enzyme, as opposed to the protein, active and nonactive, by immunocytochemistry and the precursors of the protein by in situ hybridization methods.

  16. Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

    PubMed Central

    Glaser, P; Munier, H; Gilles, A M; Krin, E; Porumb, T; Bârzu, O; Sarfati, R; Pellecuer, C; Danchin, A

    1991-01-01

    Calmodulin-activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13-24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site-directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin-binding properties of mutated proteins showed no important differences when compared to the wild-type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP-utilizing enzymes, are essential for binding the Mg(2+)-nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide-binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis. PMID:2050107

  17. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  18. The investigation of the truncated mbtA gene within the mycobactin cluster of Mycobacterium avium subspecies paratuberculosis as a novel diagnostic marker for real-time PCR.

    PubMed

    de Kruijf, Marcel; Coffey, Aidan; O'Mahony, Jim

    2017-05-01

    The inability of Mycobacterium avium subspecies paratuberculosis (MAP) to produce endogenous mycobactin in-vitro is most likely due to the presence of a truncated mbtA gene within the mycobactin cluster of MAP. The main goal of this study was to investigate this unique mbtA truncation as a potential novel PCR diagnostic marker for MAP. Novel primers were designed that were located within the truncated region and the contiguous MAP2179 gene. Primers were evaluated against non-MAP isolates and no amplicons were generated. The detection limit of this mbtA-MAP2179 target was evaluated using a range of MAP DNA concentrations, MAP inoculated faecal material and 20 MAP isolates. The performance of mbtA-MAP2179 was compared to the established f57 target. The detection limits recorded for MAP K-10 DNA and from MAP K-10 inoculated faecal samples were 0.34pg and 10(4)CFU/g respectively for both f57 and mbtA-MAP2179. A detection limit of 10(3)CFU/g was recorded for both targets, but not achieved consistently. The detection limit of MAP from inoculated faecal material was successful at 10(3)CFU/g for mbtA-MAP2179 when FAM probe real-time PCR was used. A MAP cell concentration of 10(2)CFU/g was detected successfully, but again not consistently achieved. All 20 mycobacterial isolates were successfully identified as MAP by f57 and mbtA-MAP2179. Interestingly, the mbtA-MAP2179 real-time PCR assay resulted in the formation of a unique melting curve profile that contained two melting curve peaks rather than one single peak. This melting curve phenomenon was attributed towards the asymmetrical GC% distribution within the mbtA-MAP2179 amplicon. This study investigated the implementation of the mbtA-MAP2179 target as a novel diagnostic marker and the detection limits obtained with mbtA-MAP2179 were comparable to the established f57 target, making the mbtA-MAP2179 an adequate confirmatory target. Moreover, the mbtA-MAP2179 target could be implemented in multiplex real-time PCR assays and

  19. Pituitary Adenylate Cyclase Activating Polypeptide

    PubMed Central

    Seeliger, Stephan; Buddenkotte, Jörg; Schmidt-Choudhury, Anjona; Rosignoli, Carine; Shpacovitch, Victoria; von Arnim, Ulrike; Metze, Dieter; Rukwied, Roman; Schmelz, Martin; Paus, Ralf; Voegel, Johannes J.; Schmidt, Wolfgang E.; Steinhoff, Martin

    2010-01-01

    Pituitary adenylate cyclase-activating peptide (PACAP) is an important neuropeptide and immunomodulator in various tissues. Although this peptide and its receptors (ie, VPAC1R, VPAC2R, and PAC1R) are expressed in human skin, their biological roles are unknown. Therefore, we tested whether PACAP regulates vascular responses in human skin in vivo. When injected intravenously, PACAP induced a significant, concentration-dependent vascular response (ie, flush, erythema, edema) and mediated a significant and concentration-dependent increase in intrarectal body temperature that peaked at 2.7°C. Topical application of PACAP induced marked concentration-dependent edema. Immunohistochemistry revealed a close association of PACAP-immunoreactive nerve fibers with mast cells and dermal blood vessels. VPAC1R was expressed by dermal endothelial cells, CD4+ and CD8+ T cells, mast cells, and keratinocytes, whereas VPAC2R was expressed only in keratinocytes. VPAC1R protein and mRNA were also detected in human dermal microvascular endothelial cells. The PACAP-induced change in cAMP production in these cells demonstrated VPAC1R to be functional. PACAP treatment of organ-cultured human skin strongly increased the number of CD31+ vessel cross-sections. Taken together, these results suggest that PACAP directly induces vascular responses that may be associated with neurogenic inflammation, indicating for the first time that PACAP may be a crucial vascular regulator in human skin in vivo. Antagonists to PACAP function may be beneficial for the treatment of inflammatory skin diseases with a neurogenic component. PMID:20889562

  20. Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes

    SciTech Connect

    Stadel, J.M.; Rebar, R.; Crooke, S.T.

    1987-05-01

    Desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes results in 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of ..beta..-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoproterenol- and cAMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37/sup 0/C, pH = 8.0. In both cases alkaline phosphatase treatment significantly reduced desensitization of agonist-stimulated adenylate cyclase activity by 40-60%. Similar results were obtained following alkaline phosphatase treatment of membranes from isoproterenol- and cAMP-desensitized duck erythrocytes. In addition, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with phorbol 12-mystrate 13-acetate returned adenylate cyclase activity to near control values. In all experiments inclusion of 20 mM NaPO/sub 4/ to inhibit alkaline phosphatase during treatment of membranes blocked the enzyme's effect on agonist-stimulated adenylate cyclase activity. These results demonstrate a role for phosphorylation in desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes.

  1. Globin-coupled heme containing oxygen sensor soluble adenylate cyclase in Leishmania prevents cell death during hypoxia.

    PubMed

    Sen Santara, Sumit; Roy, Jayasree; Mukherjee, Supratim; Bose, Moumita; Saha, Rina; Adak, Subrata

    2013-10-15

    Globin and adenylate cyclase play individually numerous crucial roles in eukaryotic organisms. Comparison of the amino acid sequences of globins and adenylate cyclase from prokaryotic to eukaryotic organisms suggests that they share an early common ancestor, even though these proteins execute different functions in these two kingdoms. The latest studies of biological signaling molecules in both prokaryotic and eukaryotic organisms have discovered a new class of heme-containing proteins that act as sensors. The protein of the globin family is still unknown in the trypanosomatid parasites, Trypanosome and Leishmania. In addition, globin-coupled heme containing adenylate cyclase is undescribed in the literature. Here we report a globin-coupled heme containing adenylate cyclase (HemAC-Lm) in the unicellular eukaryotic organism Leishmania. The protein exhibits spectral properties similar to neuroglobin and cytoglobin. Localization studies and activity measurements demonstrate that the protein is present in cytosol and oxygen directly stimulates adenylate cyclase activity in vivo and in vitro. Gene knockdown and overexpression studies suggest that O2-dependent cAMP signaling via protein kinase A plays a fundamental role in cell survival through suppression of oxidative stress under hypoxia. In addition, the enzyme-dependent cAMP generation shows a stimulatory as well as inhibitory role in cell proliferation of Leishmania promastigotes during normoxia. Our work begins to clarify how O2-dependent cAMP generation by adenylate cyclase is likely to function in cellular adaptability under various O2 tensions.

  2. A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

    PubMed Central

    Braga, Daniel; Hoffmeister, Dirk

    2016-01-01

    Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis. PMID:28144348

  3. Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

    SciTech Connect

    Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.; Guillon, G.; Homburger, V.

    1987-06-25

    We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher mass (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.

  4. The energy landscape of adenylate kinase during catalysis

    DOE PAGES

    Kerns, S. Jordan; Agafonov, Roman V.; Cho, Young-Jin; ...

    2015-01-12

    Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8,000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. In this paper, we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, molecular-dynamics simulations and crystallography of active complexes. We find that the Mg2+ cofactor activates two distinct molecular events: phosphoryl transfer (>105-fold) and lid opening (103-fold). In contrast, mutation of an essential active site arginine decelerates phosphoryl transfer 103-fold withoutmore » substantially affecting lid opening. Finally, our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a preorganized active site.« less

  5. The energy landscape of adenylate kinase during catalysis

    SciTech Connect

    Kerns, S. Jordan; Agafonov, Roman V.; Cho, Young-Jin; Pontiggia, Francesco; Otten, Renee; Pachov, Dimitar V.; Kutter, Steffen; Phung, Lien A.; Murphy, Padraig N.; Thai, Vu; Alber, Tom; Hagan, Michael F.; Kern, Dorothee

    2015-01-12

    Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8,000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. In this paper, we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, molecular-dynamics simulations and crystallography of active complexes. We find that the Mg2+ cofactor activates two distinct molecular events: phosphoryl transfer (>105-fold) and lid opening (103-fold). In contrast, mutation of an essential active site arginine decelerates phosphoryl transfer 103-fold without substantially affecting lid opening. Finally, our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a preorganized active site.

  6. Adenylate Energy Charge in Escherichia coli During Growth and Starvation

    PubMed Central

    Chapman, Astrid G.; Fall, Lana; Atkinson, Daniel E.

    1971-01-01

    The value of the adenylate energy charge, [(adenosine triphosphate) + ½ (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types. PMID:4333317

  7. Presence of hormonally-sensitive adenylate cyclase receptors in capillary-enriched fractions from rat cerebral cortex.

    PubMed

    Baca, G M; Palmer, G C

    1978-01-01

    The 10 000 g particulate fraction from capillary-enriched fractions isolated from rat cerebral cortex was shown to possess an adenylate cyclase highly sensitive to activation by sodium fluoride, norepinephrine, epinephrine, isoproterenol and dopamine. To a lesser extent histamine and three dopamine agonists, namely M-7 (5,6-dihydroxy-2-dimethylamino tetralin), ET-495 (methane sulfonate of pyribedil), and S-584 (metabolite of pyribedil) stimulated the enzyme preparation. The action of norepinephrine was blocked by propanolol while phenotolamine and haloperidol were relatively ineffective except at highest concentrations. Phentolamine and propanolol at only highest concentrations (10(-4) M) antagonized the action of dopamine. Haloperidol was seen to be a potent inhibitor of either dopamine- or dopamine agonist-sensitive adenylate cyclase. No effects on the enzyme were observed with methoxamine, octopamine or serotonin. These preliminary data suggest the presence of a mixed population of receptors for adenylate cyclase in rat brain capillaries.

  8. Synthetic inhibitors of adenylate kinases in the assays for ATPases and phosphokinases.

    PubMed

    Feldhau, P; Fröhlich, T; Goody, R S; Isakov, M; Schirmer, R H

    1975-09-01

    1. Procedures are given for the syntheses of alpha,omega-dinucleoside 5'-polyphosphates as inhibitors of adenylate kinases. The following order for the ability of inhibiting pig muscle adenylate kinase was observed: Ap5A greater than 1:N6-etheno-Ap5A greater than Ap6A greater than Gp5A greater than Ap4A greater than Up5A. The synthesis of adenosine tetraphosphate, the starting material for Ap5A, is also described. 2. One molecule of pig muscle adenylate kinase binds one molecule of Ap5A. The difference spectrum of Ap5A-adenylate kinase with its maximum of 5050 M-1 - cm-1 at 271 nm, as well as the fluorescence properties of 1:N6-etheno-Ap5A can be used for kinetic and binding studies. 3. The specific binding of the negatively charged Ap5A was exploited in the preparation of human muscle adenylate kinase. The enzyme was purified to homogeneity with an overall yield of 65%, the absolute value being 70 mg per kg of muscle. 4. The effect of Ap5A on adenylate kinase in extracts of various cells and cell organelles was tested. A ratio of 1:50 (mol/mol) for Ap5A to other nucleotides was used for suppressing the adenylate kinase activity in extracts of mammalian and insect skeletal muscel, of human erythrocytes and of Staphylococcus aureus. A ratio of 1:5 was found to be necessary for the adenylate kinase from tobacco leaves and spinach chloroplasts, and a ratio of 2:1 was needed for suppressing the adenylate kinase from bovine liver mitochondria, human kidney homogenate and from Escherichia coli. Ap5A appears not to be metabolized in any of the above extracts. These results indicate that Ap5A can be used for evaluating the contribution of adenylate kinase to the production of ATP fro ADP in energy-transducing systems. 5. Contaminating adenylate kinase can be inhibited by a concentration of Ap5A which does not interfere in the study of many (phospho)kinases and ATPases. The applications of Ap5A in the assay for nucleoside diphosphokinase and in the study of mechanical and

  9. WR-2721 inhibits parathyroid adenylate cyclase

    SciTech Connect

    Weaver, M.E.; Morrissey, J.; McConkey, C. Jr.; Goldfarb, S.; Slatopolsky, E.; Martin, K.J.

    1987-02-01

    WR-2721 (S-2-(3-aminopropylamino)ethylphosphorothioic acid) is a chemoprotective and radioprotective agent that has been shown to lower serum calcium in dogs and in humans. This is secondary both to impaired release of CaS from bone and diminished secretion of parathyroid hormone (PTH) from parathyroid glands. Because cAMP plays a role in the regulation of PTH secretion and WR-2721 has been shown to lower cAMP levels in radiated mouse spleen, the authors investigated the effects of WR-2721 on cAMP production in dispersed bovine parathyroid cells. Additional, they studied the adenylate cyclase in plasma membranes from normal bovine parathyroid glands after exposure to WR-2721. With parathyroid cells incubated at 0.5 mM CaS , addition of Wr-2721 in concentrations ranging from 0.02 to 2.0 mM resulted in a progressive decrease in intracellular cAMP measured by radioimmunoassay. In plasma membranes of bovine parathyroid cells a dose-dependent decrease in adenylate cyclase activity was noted. Inhibition of the cyclase was seen over a wide range of MgS concentrations. WR-2721 inhibited both basal and NaF, Gpp(NH)(, forskolin, and pertussin toxin-stimulated adenylate cyclase. These data suggest that WR-2721 inhibits the activity of parathyroid adenylate cyclase.

  10. Bordetella adenylate cyclase toxin: entry of bacterial adenylate cyclase into mammalian cells.

    PubMed

    Confer, D L; Slungaard, A S; Graf, E; Panter, S S; Eaton, J W

    1984-01-01

    We have identified an adenylate cyclase toxin in urea extracts and culture supernatant fluids of Bordetella pertussis (2). The ability of this toxin and the lack of a strong correlation between its activity and adenylate cyclase activity found in urea extracts suggest that it is an oligomer of readily dissociable subunits. The mechanism by which Bordetella adenylate cyclase toxin interacts with target cells is unknown, but polyvalent cations are necessary. Neutrophils exposed to the toxin acquire a 39,000 Mr protein that can also be photoaffinity labeled with 32P-ATP. We anticipate that this protein will prove to be a catalytic component of Bordetella adenylate cyclase toxin. Susceptible cells exposed to Bordetella adenylate cyclase toxin are functionally aberrant. In phagocytes, decreased bactericidal capacity may be important in the pathogenesis of human whooping cough and other Bordetella infections occurring in domestic animals. The effects of the toxin on neoplastic cells may offer new insights into the factors controlling their growth and differentiation. Bordetella adenylate cyclase toxin is a unique bacterial product. Further purification and characterization of this toxin will add to our understanding of cell-protein interactions and pathogen-host relationships.

  11. Effect of mitomycin C on the activation of adenylate cyclase in rat ascites hepatoma AH130 cells.

    PubMed

    Miyamoto, K; Matsunaga, T; Sanae, F; Koshiura, R

    1986-09-01

    Isoproterenol (IPN)-stimulated activity of adenylate cyclase was enhanced in a dose-dependent manner by exposure of AH130 cells to mitomycin C (MMC). The enhancement was also observed in prostaglandin E1-, guanine nucleotide analog-, NaF-, cholera toxin- and forskolin-stimulated activities of the enzyme but not in manganese-stimulated activity. In addition, even when the cells pretreated with islet-activating protein were exposed to MMC, IPN-stimulated activity of adenylate cyclase was enhanced. Anaerobic exposure of AH130 cells to MMC somewhat inhibited IPN-stimulated activity of adenylate cyclase in contrast with aerobic exposure. Exposure of cells to adriamycin also caused enhancement of IPN-stimulated activity of adenylate cyclase but exposure to nitrogen mustard inhibited the enzyme stimulation by IPN. The enhancing effect of MMC was lost by the combined treatment with alpha-tocopherol. From these results, it was shown that MMC modulated the activity of adenylate cyclase, probably through alterations in membrane structure.

  12. Topographic separation of adenylate cyclase and hormone receptors in the plasma membrane of toad erythrocyte ghosts

    PubMed Central

    Sahyoun, N.; Hollenberg, M. D.; Bennett, V.; Cuatrecasas, P.

    1977-01-01

    Brief sonication of whole erythrocyte plasma membranes (ghosts) from toads at 4° does not inactivate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] or destroy the receptor binding properties of hydroxybenzylpindolol or insulin. The hormonal (but not the fluoride-induced) stimulation of this enzyme is, however, lost. Fractionation of the small, resealed membrane fragments (vesicles) on discontinuous sucrose gradients results in the separation of vesicle populations differing grossly in size and protein composition. In addition, the distribution of the β-adrenergic receptor, an insulin binding site, and adenylate cyclase among these vesicles fractions differs. The pattern of distribution of these functional structures can be altered differentially by manipulations of the ghosts before sonication. For example, brief preincubation with isoproterenol leads to a change in the relative distribution of β-receptor (but not adenylate cyclase) among the various vesicle fractions; this effect is not obtained with β-receptor antagonists, which block the isoproterenol effect. Exposure of the ghosts to different temperatures, changes in the divalent cation composition of the medium, or the addition of ATP also leads to changes in the distribution of surface markers of the subsequently formed vesicles. The results indicate gross asymmetries in the distribution of protein components within the plane of the membrane and raise important questions regarding the manner whereby functionally related and coupled components, such as hormone receptors and adenylate cyclase, interact. Images PMID:197522

  13. Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides.

    PubMed Central

    Sulakhe, P V; Leung, N L; Arbus, A T; Sulakhe, S J; Jan, S H; Narayanan, N

    1977-01-01

    1. GTP and GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) were observed to increase the stimulation of neural adenylate cyclase by dopamine (3,4-dihydroxyphenethylamine) and noradrenaline. 2. GMP-P(NH)P had a biphasic effect on the enzyme activity. 3. Preincubation of membranes with GMP-P(NH)P activated the enzyme by a process dependent on time and temperature. Catecholamines increased the speed and the extent of this activation. 4. Membrane fractions contained high- and low-affinity sites for GMP-P(NH)P binding: this binding was due to protein(s) of the membrane preparations. 5. Low-affinity-site binding of GMP-P(NH)P appeared to be related to the stimulatory effect on the adenylate cyclase activity. PMID:18147

  14. Linkage between Fitness of Yeast Cells and Adenylate Kinase Catalysis

    PubMed Central

    Tükenmez, Hasan; Magnussen, Helge Magnus; Kovermann, Michael; Byström, Anders; Wolf-Watz, Magnus

    2016-01-01

    Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies. PMID:27642758

  15. Antitubercular Nucleosides that Inhibit Siderophore Biosynthesis: SAR of the Glycosyl Domain

    PubMed Central

    Somu, Ravindranadh V.; Wilson, Daniel; Bennett, Eric M.; Boshoff, Helena; Celia, Laura; Beck, Brian; Barry, Clifton E.; Aldrich, Courtney C.

    2008-01-01

    Tuberculosis (TB) is the leading cause of infectious disease mortality in the world by a bacterial pathogen. We previously demonstrated that a bisubstrate inhibitor of the adenylation enzyme MbtA, which is responsible for the second step of mycobactin biosynthesis, exhibited potent antitubercular activity. Here we systematically investigate the structure activity relationships of the bisubstrate inhibitor glycosyl domain resulting in the identification of a carbocyclic analogue that possesses a KIapp value of 2.3 nM and MIC99 values of 1.56 μM against M. tuberculosis H37Rv. The SAR data suggest the intriguing possibility that the bisubstrate inhibitors utilize a transporter for entry across the mycobacterial cell-envelope. Additionally, we report improved conditions for the expression of MbtA and biochemical analysis demonstrating that MbtA follows a random sequential enzyme mechanism for the adenylation half-reaction. PMID:17181146

  16. Mechanisms of nonhormonal activation of adenylate cyclase based on target analysis

    SciTech Connect

    Verkman, A.S.; Ausiello, D.A.; Jung, C.Y.; Skorecki, K.L.

    1986-08-12

    Radiation inactivation was used to examine the mechanism of activation of adenylate cyclase in the cultured renal epithelial cell line LLC-PK1 with hormonal (vasopressin) and nonhormonal (GTP, forskolin, fluoride, and chloride) activating ligands. Intact cells were frozen, irradiated at -70 degrees C (0-14 Mrad), thawed, and assayed for adenylate cyclase activity in the presence of activating ligands. The ln (adenylate cyclase activity) vs. radiation dose relation was linear (target size 162 kDa) for vasopressin- (2 microM) stimulated activity and concave downward for unstimulated (10 mM Mn/sup 2 +/), NaF- (10 mM) stimulated, and NaCl- (100 mM) stimulated activities. Addition of 2 microM vasopressin did not alter the ln activity vs. dose relation for NaF- (10 mM) stimulated activity. The dose-response relations for adenylate cyclase activation and for transition in the ln activity vs. dose curve shape were measured for vasopressin and NaF. On the basis of our model for adenylate cyclase subunit interactions reported previously (Verkman, A. S., Skorecki, K. L., and Ausiello, D. A. (1986) Am. J. Physiol. 260, C103-C123) and of new mathematical analyses, activation mechanisms for each ligand are proposed. In the unstimulated state, equilibrium between alpha beta and alpha + beta favors alpha beta; dissociated alpha binds to GTP (rate-limiting step), which then combines with the catalytic (C) subunit to form active enzyme. Vasopressin binding to receptor provides a rapid pathway for GTP binding to alpha. GTP and its analogues accelerate the rate of alpha GTP formation. Forskolin inhibits the spontaneous deactivation of activated C. Activation by fluoride may occur without alpha beta dissociation or GTP addition through activation of C by an alpha beta-F complex.

  17. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

    2011-02-18

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  18. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  19. Serotonin-Sensitive Adenylate Cyclase in Neural Tissue and Its Similarity to the Serotonin Receptor: A Possible Site of Action of Lysergic Acid Diethylamide

    PubMed Central

    Nathanson, James A.; Greengard, Paul

    1974-01-01

    An adenylate cyclase (EC 4.6.1.1) that is activated specifically by low concentrations of serotonin has been identified in homogenates of the thoracic ganglia of an insect nervous system. The activation of this enzyme by serotonin was selectively inhibited by extremely low concentrations of D-lysergic acid diethylamide (LSD), 2-bromo-LSD, and cyproheptadine, agents which are known to block certain serotonin receptors in vivo. The inhibition was competitive with respect to serotonin, and the calculated inhibitory constant of LSD for this serotonin-sensitive adenylate cyclase was 5 nM. The data are consistent with a model in which the serotonin receptor of neural tissue is intimately associated with a serotonin-sensitive adenylate cyclase which mediates serotonergic neurotransmission. The results are also compatible with the possibility that some of the physiological effects of LSD may be mediated through interaction with serotonin-sensitive adenylate cyclase. PMID:4595572

  20. Adenylation-Dependent Conformation and Unfolding Pathways of the NAD+-Dependent DNA Ligase from the Thermophile Thermus scotoductus

    PubMed Central

    Georlette, Daphné; Blaise, Vinciane; Bouillenne, Fabrice; Damien, Benjamin; Thorbjarnardóttir, Sigridur H.; Depiereux, Eric; Gerday, Charles; Uversky, Vladimir N.; Feller, Georges

    2004-01-01

    In the last few years, an increased attention has been focused on NAD+-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium. However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD+-dependent DNA ligases are understood. From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD+-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation. Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme. These data support an induced “open-closure” process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA. These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA. In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability. Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states. Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase. These data provide further insights into the properties of partially folded intermediates. PMID:14747344

  1. Demonstration of phosphoryl group transfer indicates that the ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) exhibits adenylate kinase activity.

    PubMed

    Randak, Christoph O; Ver Heul, Amanda R; Welsh, Michael J

    2012-10-19

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5'-monophosphate (AMP), CFTR Cl(-) channel function is coupled to adenylate kinase activity (ATP+AMP <==> 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the (32)P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase.

  2. Influence of volatile anesthetics on muscarinic receptor adenylate cyclase coupling in brain and heart

    SciTech Connect

    Anthony, B.L.

    1988-01-01

    In the present study, the influence of four volatile anesthetics (enflurane, isoflurane, diethyl ether, and chloroform) on (1) muscarinic receptor binding parameters and (2) muscarnic regulation of adenylate cyclase activity was examined using membranes isolated from rat brain and heart. Membranes were equilibrated with each of the four anesthetics for 30 minutes and then during the binding assay. The data obtained can be summarized as follows: (1) volatile anesthetics increased receptor affinity for a radiolabeled antagonists, ({sup 3}H)N-methylscopolamine (({sup 3}H)MS), by decreasing its rate of dissociation in brain stem, but not in cardiac, membranes, (2) volatile anesthetics decreased high affinity ({sup 3}H)Oxotremorine-M binding, (3) volatile anesthetics depressed or eliminated the guanine nucleotide sensitivity of agonist binding. The influence of volatile anesthetics on muscarinic regulation of adenylate cyclase enzyme activity was studied using {alpha}({sup 32}P)ATP as the substrate.

  3. Conformational Dynamics of a Ligand-Free Adenylate Kinase

    PubMed Central

    Song, Hyun Deok; Zhu, Fangqiang

    2013-01-01

    Adenylate kinase (AdK) is a phosphoryl-transfer enzyme with important physiological functions. Based on a ligand-free open structure and a ligand-bound closed structure solved by crystallography, here we use molecular dynamics simulations to examine the stability and dynamics of AdK conformations in the absence of ligands. We first perform multiple simulations starting from the open or the closed structure, and observe their free evolutions during a simulation time of 100 or 200 nanoseconds. In all seven simulations starting from the open structure, AdK remained stable near the initial conformation. The eight simulations initiated from the closed structure, in contrast, exhibited large variation in the subsequent evolutions, with most (seven) undergoing large-scale spontaneous conformational changes and approaching or reaching the open state. To characterize the thermodynamics of the transition, we propose and apply a new sampling method that employs a series of restrained simulations to calculate a one-dimensional free energy along a curved pathway in the high-dimensional conformational space. Our calculated free energy profile features a single minimum at the open conformation, and indicates that the closed state, with a high (∼13 kcal/mol) free energy, is not metastable, consistent with the observed behaviors of the unrestrained simulations. Collectively, our simulations suggest that it is energetically unfavorable for the ligand-free AdK to access the closed conformation, and imply that ligand binding may precede the closure of the enzyme. PMID:23861846

  4. Studies on adenosine triphosphate transphosphorylases. Human isoenzymes of adenylate kinase: isolation and physicochemical comparison of the crystalline human ATP-AMP transphosphorylases from muscle and liver.

    PubMed

    Kuby, S A; Fleming, G; Frischat, A; Cress, M C; Hamada, M

    1983-02-10

    Procedures are described for the isolation, in crystalline form, of the adenylate kinases from autopsy samples of human muscle and from human liver. Weight average molecular weights were determined by sedimentation equilibrium to be 22,000 (+/- 700) and 25,450 (+/- 160) for the human muscle and liver isoenzymes, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their molecular weights were estimated to be 21,700 and 26,500 for the muscle and liver enzymes, respectively. Both isoenzymes are accordingly monomeric proteins in their native state. Amino acid analyses are reported here for the normal human liver, calf liver, and rabbit liver adenylate kinases and compared with the normal human muscle, calf muscle, and rabbit muscle myokinases. The liver types as a group and the muscle types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, half-cystine, and the presence of tryptophan in the liver enzymes. The normal human liver adenylate kinase, as isolated in this report, has proved to be similar in its properties, if not identical, to the adenylate kinase isolated directly from human liver mitochondria (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S. A. (1982) J. Biol. Chem. 257, 13120-13128). Therefore, the liver-type adenylate kinase may be considered a mitochondrial type.

  5. High level expression of chicken muscle adenylate kinase in Escherichia coli.

    PubMed

    Tanizawa, Y; Kishi, F; Kaneko, T; Nakazawa, A

    1987-05-01

    Chicken muscle adenylate kinase was produced in a large amount in Escherichia coli cells harboring an expression plasmid, pKK-cAKl-1. The plasmid was constructed by placing the cDNA sequence for chicken muscle adenylate kinase after the tac promoter. After induction by isopropyl-beta-D-thiogalactopyranoside, the enzyme protein amounted to about 10% of the bacterial proteins. The enzyme was readily purified in two steps by using phosphocellulose and Sephadex G-100 columns. The apparent molecular weight of the enzyme produced in E. coli was estimated to be 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in agreement with the value deduced from the cDNA sequence. Ten amino acids in the NH2-terminal region were determined, and were identical with the sequence deduced from the cDNA sequence except that the terminal methionine was absent. Michaelis constants for ATP, ADP, and AMP of the enzyme thus synthesized were essentially identical to those determined with the enzyme in crude extracts of chicken skeletal muscle.

  6. Ontogenic development of antidiuretic hormone receptors in rat kidney: comparison of hormonal binding and adenylate cyclase activation.

    PubMed

    Rajerison, R M; Butlen, D; Jard, S

    1976-03-01

    The development of adenylate cyclase responsiveness to vasopressin and parathyroid hormone was studied using membrane fractions prepared from medullo-papillary and cortical portions of kidneys of 2-46-day-old rats. The development of vasopressin binding capacity was followed on the same preparations, using [3H]vasopressin. The characteristics of medullo-papillary adenylate cyclase response to vasopressin were identical in young and adult control animals as regards apparent Km values for [Lys8]vasopressin (3 X 10(-8) M), specificity towards the natural neurohypophysial peptides and the effects of Mg2+. However, the magnitude of maximal enzyme activation by vasopressin was much lower in very young than adult animals. Accordingly vasopressin responsiveness increased sharply between the 10th and 25th days but the magnitude of the maximal response only reached the adult value between the 30th and 45th days after birth. During both periods basal adenylate cyclase activity was almost independent of age. Specific vasopressin binding sites were detected on kidney medullo-papillary membranes from young animals. Vasopressin binding capacity and adenylate cyclase responsiveness to the hormone followed similar development patterns. However, the appearance of specific binding sites slightly preceded the onset of adenylate cyclase responsiveness. Basal cortical adenylate cyclase activity/mg protein was 12 times higher in 2-day-old rats than in the adult controls. It dropped with age but only fell to the adult value between the 25th and the 35th days after birth. For the youngest animals tested (2 days old), the increase in activity due to parathyroid hormone was about half the increase measured in adults, and gradually rose to about 75% of the adult response between the 2nd and 46th days after birth. Apparent Km values for parathyroid hormone were identical in young and adult animals (3.2 and 3.0 U/ml, respectively).

  7. Antineoplastic effects of Bordetella pertussis adenylate cyclase.

    PubMed

    Slungaard, A; Confer, D L; Jacob, H S; Eaton, J W

    1983-01-01

    Urea extracts of B. pertussis, but not B. bronchiseptica, cause large and sustained intracellular cAMP elevation in several neoplastic cell lines. These cAMP elevations are associated with growth inhibition (HL-60, Friend erythroleukemia) and a phenotypic change/differentiation (HL-60, L1210). B. pertussis extract injections prolong survival of L1210 tumor-bearing mice. Pretreatment of L1210 cells with B. pertussis extract both delays mortality and induces growth of solid tumors instead of ascites in subsequently inoculated mice. We conclude that B. pertussis adenylate cyclase is capable of invading a variety of neoplastic cells to catalyze the intracellular formation of large amounts of cAMP. These cAMP elevations are durable and promote growth arrest, differentiation, or phenotypic alterations reflected in altered biologic behavior. B. pertussis adenylate cyclase should prove to be a useful tool for manipulating cAMP levels in neoplastic cells to elucidate the role of cAMP in malignant transformation.

  8. Adenylate Cyclase Activity Not Found in Soybean Hypocotyl and Onion Meristem 1

    PubMed Central

    Yunghans, Wayne N.; Morré, D. James

    1977-01-01

    Tissue, homogenates, and purified cell fractions prepared from hypocotyls of a dicot, soybean (Glycine max), and meristematic tissue of a monocot, onion (Allium cepa), were examined critically for evidence of adenylate cyclase activity. Three assay methods were used: chemical analysis, isotope dilution analysis, and enzyme cytochemistry. In both crude extracts or whole tissue, as well as purified membranes, with or without auxin, no adenylate cyclase was detected by any of the three methods. For plasma membranes, the specific activity was less than 1/40 or 1/25,000 that of rat liver plasma membranes, depending on the assay procedure, i.e. below the limits of detection. Using comparable methods, we could detect neither cyclic adenosine 3′:5′-monophosphate nor the phosphodiesterase responsible for its degradation in either purified membranes or homogenates. The results suggest that hormone responses in plants are not generally mediated by a mechanism involving the obligate production of cyclic adenosine 3′:5′-monophosphate by a plasma membrane associated adenylate cyclase. Images PMID:16660026

  9. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    SciTech Connect

    Niles, L.P.; Hashemi, F. )

    1990-12-01

    1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, ({sup 125}I)iodomelatonin, was examined using an incubation temperature (30{degree}C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing ({sup 125}I)iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.

  10. Equilibration of adenylates in the mitochondrial intermembrane space maintains respiration and regulates cytosolic metabolism.

    PubMed

    Igamberdiev, Abir U; Kleczkowski, Leszek A

    2006-01-01

    Adenylate kinase (AK) uses one each of Mg-complexed and free adenylates as substrates in both directions of its reaction. It is very active in the mitochondrial intermembrane space (IMS), but is absent from the mitochondrial matrix where low [ADP] upon intensive respiration limits the respiratory rate. AK activity in the IMS is linked to ATP/ADP exchange across the inner mitochondrial membrane by using ATP (imported from the matrix) and AMP as substrates, the latter provided by apyrase and other AMP-generating reactions. The ADP formed by AK is exported to the matrix (in exchange for ATP), providing a mechanism for regeneration of ADP during respiration. From the AK equilibrium, and taking pH values characteristic of subcellular compartments, [Mg2+] in the IMS is calculated as 0.4-0.5 mM and in the cytosol as 0.2-0.3 mM, whereas the MgATP:MgADP ratio in the IMS and cytosol is 6-9 and 10-15, respectively. These represent optimal conditions for transport of adenylates (via the maintenance of an ATPfree:ADPfree ratio close to 1) and mitochondrial respiratory rates (via the maintenance of submillimolar [ADPfree] in the IMS). This, in turn, has important consequences for mitochondrial and cytosolic metabolism, including regulation of the protein phosphorylation rate (via changes in the MgATP:AMPfree ratio) and allosteric regulation of mitochondrial and cytosolic enzymes. Metabolomic consequences are discussed in connection with the calculation of metabolic fluxes from subcompartmental distributions of total adenylates and Mg2+.

  11. Enhanced serotonin-stimulated adenylate cyclase activity in membranes from adult guinea pig hippocampus.

    PubMed

    Shenker, A; Maayani, S; Weinstein, H; Green, J P

    1983-05-16

    We have developed an assay for serotonin (5-HT) stimulation of adenylate cyclase activity in membranes from adult guinea pig hippocampus. The response to 5-HT is concentration-dependent, with an EC50 of 0.01 microM, a shallow slope, and mean maximal stimulation of 90% over basal activity. The response to 5-HT is GTP-dependent and additive to the maximal stimulation by histamine. Micromolar concentrations of the known 5-HT receptor agonists, tryptamine and 5-methoxytryptamine, also stimulate cAMP production in this system, and their effect is not additive to that elicited by a maximal concentration of 5-HT. These results are consistent with the hypothesis that the response to 5-HT is elicited through a distinct receptor coupled to adenylate cyclase; the magnitude and the reproducibility of the 5-HT response in this system should make it useful for receptor classification. To examine the effect of prior exposure to endogenous 5-HT on the responsiveness of the system, we assayed 5-HT stimulation of enzyme activity in membranes prepared from animals 25-27 hrs after treatment with a single injection of reserpine (5 mg/kg, i.p.). The mean maximal stimulation of adenylate cyclase by 5-HT was increased to 150% over basal activity with no effect on the EC50 or slope of the 5-HT concentration-response curve. Reserpine pretreatment did not affect basal activity or histamine-stimulated adenylate cyclase activity. These results are discussed in the context of a hypothesis that endogenous 5-HT normally exerts a desensitizing effect on its receptors in situ.

  12. Study into the kinetic properties and surface attachment of a thermostable adenylate kinase

    PubMed Central

    Hathaway, H.J.; Sutton, J.M.; Jenkins, A.T.A.

    2015-01-01

    A thermostable adenylate kinase (tAK) has been used as model protein contaminant on surfaces, so used because residual protein after high temperature wash steps can be detected at extremely low concentrations. This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination. Current methods utilise non-covalent (physisorbtion) of tAK to surfaces, but this can be relatively easily removed. In this study, the covalent binding of tAK to surfaces was studied to provide an alternative model for surface contamination. Kinetic analysis showed that the efficiency of the enzyme expressed as the catalytic rate over the Michaelis constant (kcat/KM) increased from 8.45±3.04 mM−1 s−1 in solution to 32.23±3.20 or 24.46±4.41 mM−1 s−1 when the enzyme was immobilised onto polypropylene or plasma activated polypropylene respectively. Maleic anhydride plasma activated polypropylene showed potential to provide a more robust challenge for washing processes as it retained significantly higher amounts of tAK enzyme than polypropylene in simple washing experiments. Inhibition of the coupled enzyme (luciferase/luciferin) system used for the detection of adenylate kinase activity, was observed for a secondary product of the reaction. This needs to be taken into consideration when using the assay to estimate cleaning efficacy. PMID:26339684

  13. Energetics and Structural Characterization of the large-scale Functional Motion of Adenylate Kinase

    NASA Astrophysics Data System (ADS)

    Formoso, Elena; Limongelli, Vittorio; Parrinello, Michele

    2015-02-01

    Adenylate Kinase (AK) is a signal transducing protein that regulates cellular energy homeostasis balancing between different conformations. An alteration of its activity can lead to severe pathologies such as heart failure, cancer and neurodegenerative diseases. A comprehensive elucidation of the large-scale conformational motions that rule the functional mechanism of this enzyme is of great value to guide rationally the development of new medications. Here using a metadynamics-based computational protocol we elucidate the thermodynamics and structural properties underlying the AK functional transitions. The free energy estimation of the conformational motions of the enzyme allows characterizing the sequence of events that regulate its action. We reveal the atomistic details of the most relevant enzyme states, identifying residues such as Arg119 and Lys13, which play a key role during the conformational transitions and represent druggable spots to design enzyme inhibitors. Our study offers tools that open new areas of investigation on large-scale motion in proteins.

  14. Heterologous desensitization of adenylate cyclase from pigeon erythrocytes under the action of the catalytic subunit of cAMP-dependent protein kinase

    SciTech Connect

    Popov, K.M.; Bulargina, T.V.; Severin, E.S.

    1985-09-20

    Preincubation of the plasma membranes from pigeon erythrocytes with the catalytic subunit of cAMP-dependent protein kinase leads to desensitization of adenylate cyclase of the erythrocytes. The adenylate cyclase activity, measured in the presence of 10 ..mu..M isoproterenol and 50 ..mu..M GTP-..gamma..-S, is decreased by 40% in 10 min of incubation, while the activity in the presence of 50 ..mu..M GTP-..gamma..-S is decreased by 35% in 20 min. The decrease in the adenylate cyclase activity is due to an increase in the lag phase of activation of the enzyme in the presence of a GTP analog stable to hydrolysis and a decrease in the activity in the steady-state phase of activation. Heterologous desensitization of adenylate cyclase under the action of cAMP-dependent protein kinase is coupled with a decrease in the number of ..beta..-adrenoreceptors capable of passing into a state of high affinity for antagonists in the absence of guanylic nucleotides. The influence of the catalytic subunit on adenylate cyclase entirely models the process of desensitization of the enzyme absorbed in the influence of isoproterenol or cAMP on erythrocytes.

  15. Cellular interactions uncouple beta-adrenergic receptors from adenylate cyclase.

    PubMed

    Ciment, G; de Vellis, J

    1978-11-17

    C6 glioma cells and B104 neuroblastoma cells both possess adenylate cyclase activity, but only C6 cells have beta-adrenergic receptors. However, when cocultured with B104 cells, C6 cells show a marked decrease in their ability to accumulate adenosine 3', 5'-monophosphate upon stimulation with beta receptor agonists. Since both beta receptors and cholera toxin-stimulated adenylate cyclase activities are present in C6/B104 cocultures, we conclude that the beta receptor/adenylate cyclase transduction mechanism in cocultured C6 cells is uncoupled.

  16. Identification of a haem domain in human soluble adenylate cyclase

    PubMed Central

    Middelhaufe, Sabine; Leipelt, Martina; Levin, Lonny R.; Buck, Jochen; Steegborn, Clemens

    2012-01-01

    The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle. PMID:22775536

  17. Adenylate kinase locus 1 polymorphism and feto-placental development.

    PubMed

    Fulvia, Gloria-Bottini; Antonio, Pietroiusti; Anna, Neri; Patrizia, Saccucci; Ada, Amante; Egidio, Bottini; Andrea, Magrini

    2011-12-01

    Recently our group has found that the correlation between birth weight and placental weight - an index of a balanced feto-placental unit development - is influenced by genetic factors. Since adenylate kinase locus 1 (AK₁) is a polymorphic enzyme that plays an important role in the synthesis of nucleotides required for many metabolic functions, we have investigated the possible role of its genetic variability in the correlation between birth weight and placental weight. 342 consecutive healthy newborn infants from the population of Rome (Italy) and 286 puerperae from another population from Central Italy were studied. The correlation coefficient between birth weight and placental weight is much higher in infants with low activity AK₁2-1 phenotype than in those with high activity AK₁1 phenotype. The difference between AK₁ and AK₁2-1 is well marked only in newborns with a gestational age greater than 38 weeks and it is not influenced by sex, maternal age and maternal smoking. A similar pattern is observed with maternal AK₁ phenotype. These results suggest that the difference in enzymatic activity between AK₁ phenotypes influencing the equilibrium among ATP, ADP, AMP and adenosine could have an important role in a balanced development of feto-placental unit. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli.

    PubMed

    Liu, Xinxin; Huang, Anliang; Luo, Dan; Liu, Haipeng; Han, Huzi; Xu, Yang; Liang, Peng

    2015-05-01

    The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E.coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. NMR studies of the AMP-binding site and mechanism of adenylate kinase.

    PubMed

    Fry, D C; Kuby, S A; Mildvan, A S

    1987-03-24

    NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase [Fry, D. C., Kuby, S. A., & Mildvan, A. S. (1985) Biochemistry 24, 4680-4694]. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr3+AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T1 method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high anti-glycosyl torsional angle (chi = 110 +/- 10 degrees), a 3'-endo,2'-exo ribose pucker (delta = 105 +/- 10 degrees), and gauche-trans orientations about the C4'-C5' bond (gamma = 180 +/- 10 degrees) and the C5'-O5' bond (beta = 170 +/- 20 degrees). The distance from Cr3+ to the phosphorus of AMP is 5.9 +/- 0.3 A, indicating a reaction coordinate distance of approximately 3 A, which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near (less than or equal to 4 A from) Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., & Manship, M. (1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F., Maruyama, M., Tanizawa, Y

  20. Adenylate cyclase and G-proteins as a signal transfer system in the guinea pig inner ear.

    PubMed

    Koch, T; Zenner, H P

    1988-01-01

    In many eukaryotic cells G-proteins play a key role in signal transduction through outer cell membranes. To study this pathway in the auditory organ of mammals we examined tissue preparations from the stria vascularis and the organ of Corti from the guinea pig inner ear. The activity of adenylate cyclase was measured by stimulation at the site of the enzyme, the hormone receptors and the modulating G-proteins. In the organ of Corti we found a low enzyme activity in all cochlear turns. The stria vascularis, however, showed a constant high concentration of beta 2-adrenergic receptors and of stimulating G-proteins in all cochlear turns. In contrast, the activity of the enzyme increased from the apical to the basal turn. Adenylate cyclase could be stimulated or inhibited in a concentration-dependent manner by drugs selectively effecting the G-proteins. Our results suggest a structure of the adenylate cyclase complex in the inner ear similar to other organs. Pathophysiological correlations to hearing loss associated with pseudohypoparathyroidism are discussed.

  1. Iodide-induced inhibition of adenylate cyclase activity in horse and dog thyroid.

    PubMed

    Cochaux, P; Van Sande, J; Swillens, S; Dumont, J E

    1987-12-30

    The characteristics of the iodide-induced inhibition of cyclic AMP accumulation in dog thyroid slices have been previously described [Van Sande, J., Cochaux, P. and Dumont, J. E. (1985) Mol. Cell. Endocrinol. 40, 181-192]. In the present study we investigated the characteristics of the iodide-induced inhibition of adenylate cyclase activity in dog and horse thyroid. The inhibition of cyclic AMP accumulation by iodide in stimulated horse thyroid slices was similar to that observed in dog thyroid slices. The inhibition was observed in slices stimulated by thyroid-stimulating hormone, cholera toxin and forskolin. Increasing the concentration of the stimulators did not overcome the iodide-induced inhibition. Adenylate cyclase activity, assayed in crude homogenates or in plasma-membrane-containing particulates (100,000 x g pellets), was lower in homogenates or in particulates prepared from iodide-treated slices than from control slices. This inhibition was observed on the cyclase activity stimulated by forskolin, fluoride or guanosine 5'-[beta, gamma-imino]triphosphate, but also on the basal activity. It was relieved when the homogenate was prepared from slices incubated with iodide and methimazole. Similar results were obtained with dog thyroid. The inhibition persisted when the particulate fraction was washed three times during 1 h at 100,000 x g, in the presence of bovine serum albumin or increasing concentration of KCl. It was similar whatever the duration of the cyclase assay, in a large range of protein concentration. These results indicate that a stable modification of adenylate cyclase activity, closely related to the plasma membrane, was induced when slices were incubated with iodide. Iodide inhibition did not modify the affinity of adenylate cyclase for its substrate (MgATP), but induced a decrease of the maximal velocity of the enzyme. The percentage inhibition was slightly decreased when Mg2+ concentration increased, and markedly decreased when Mn2

  2. Identification of valine/leucine/isoleucine and threonine/alanine/glycine proton-spin systems of Escherichia coli adenylate kinase by selective deuteration and selective protonation.

    PubMed Central

    Bock-Möbius, I; Brune, M; Wittinghofer, A; Zimmermann, H; Leberman, R; Dauvergne, M T; Zimmermann, S; Brandmeier, B; Rösch, P

    1991-01-01

    Adenylate kinase from two types of Escherichia coli strains, a wild-type and a leucine-auxotrophic strain, was purified. On the one hand, growing the leucine-auxotrophic bacteria on a medium containing deuterated leucine yielded E. coli adenylate kinase with all leucine residues deuterated. On the other hand, by growing the wild-type bacteria on deuterated medium with phenylalanine, threonine and isoleucine present as protonated specimens, 80% randomly deuterated enzyme with protonated phenylalanine, threonine and isoleucine residues could be prepared. Use of these proteins enabled identification of the spin systems of these amino acid residues in the n.m.r. spectra of the protein. PMID:1991031

  3. Ultraviolet radiation augments epidermal beta-adrenergic adenylate cyclase response

    SciTech Connect

    Iizuka, H.; Kajita, S.; Ohkawara, A.

    1985-05-01

    Pig skin was irradiated in vivo with fluorescent sunlamp tubes (peak emission at 305 nm). A significant increase in epidermal beta-adrenergic adenylate cyclase response was observed as early as 12 h following 1-2 minimum erythema doses (MEDs) UVB exposure, which lasted at least 48 h. The augmentation of adenylate cyclase response was relatively specific to the beta-adrenergic system and there was no significant difference in either adenosine- or histamine-adenylate cyclase response of epidermis. The increased beta-adrenergic adenylate cyclase response was less marked at higher doses of UVB exposure (5 MEDs); in the latter condition, a significant reduction in adenosine- or histamine-adenylate cyclase response was observed. There was no significant difference in either low- or high-Km cyclic AMP phosphodiesterase activity between control and UVB-treated skin at 1-2 MEDs. These data indicate that the epidermal adenylate cyclase responses are affected in vivo by UVB irradiation, which might be a significant regulatory mechanism of epidermal cyclic AMP systems.

  4. Ethrel (Ethylene Releaser)-Induced Increases in the Adenylate Pool and Transtonoplast ΔpH within Hevea Latex Cells

    PubMed Central

    Amalou, Zakia; Bangratz, Jacques; Chrestin, Hervé

    1992-01-01

    The treatment of rubber tree (Hevea brasiliensis) bark with chloro-2-ethyl phosphonic acid (ethrel), an ethylene-releasing chemical, induced, after a lag period of 13 to 21 hours, a marked increase in the total adenine nucleotides (essentially ATP and ADP) of latex cells. This rise in the latex adenylate pool was concomitant with a marked decrease in the [ATP]/[ADP] ratio without significant changes in the adenylate energy charge. The apparent equilibrium constant for the adenylate kinase, which appeared to behave as a key enzyme in maintaining the adenylate energy charge in the latex, was considerably reduced, probably as a consequence of the alkalinization of the latex cytosol induced by the treatment with ethrel. To reduce the “sink effect” and activation of the metabolism induced in Hevea bark by regular tapping, the latex was collected by micropuncture (few drops) at increasing distance (5-50 centimeters) above and below an ethrel-treated area on the virgin bark of resting trees. The effect of ethrel was shown to spread progressively along the trunk. The increase in the adenylate pool (essentially ATP) was detectable as early as 24 hours after the bark treatment and was maximum after 6 or 8 days, 5 centimeters as well as 50 centimeters above and below the stimulated bark ring. The correlative vacuolar acidification and cytosolic alkalinization, i.e. the increase in the transtonoplast ΔpH, induced in the latex cells by ethrel were shown to be concomitant with the rise in ATP content of the latex. This suggests that the tonoplast H+-pumping ATPase, which catalyzes vacuolar acidification in the latex, is directly and essentially under the control of the availability of its substrate (i.e. ATP) in the latex. The results are discussed in relation to energy-dependent activation of metabolism, and increased rubber production, as induced by the stimulation of rubber trees with ethrel. PMID:16668787

  5. Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances.

    PubMed

    Howell, Stanley C; Richards, David H; Mitch, William A; Wilson, Corey J

    2015-10-16

    Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study, we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary structure, protein hydrodynamics, and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance, namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites.

  6. Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances

    PubMed Central

    Howell, Stanley C.; Richards, David H.; Mitch, William A.; Wilson, Corey J.

    2016-01-01

    Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections, and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary-structure, protein hydrodynamics and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance. Namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites. PMID:26266833

  7. Adenylate cyclase regulation in the spermatogenic cell plasma membrane: Modulating effects of TPA and TCDD

    SciTech Connect

    Beebe, L.E.

    1989-01-01

    This research was designed to compare the effects of TPA, a phorbol ester, and TCDD in a spermatogenic cell population, a target of TCDD toxicity. Membrane-bound adenylate cyclase activity was used an index of membrane function, and was quantified by the amount of {sup 32}P-cAMP formed from {sup 32}P-ATP following chromatographic separation. Exposure to male germ cells in-vitro to TPA and TCDD followed by direct measurement of enzyme activity was used to investigate the potential of each agent to perturb membrane function. TPA and TCDD consistently inhibited adenylate cyclase activity at the levels of G{sub s}-catalytic unit coupling and hormone-receptor activation, as measured by the stimulation of enzyme activity by concomitant addition of forskolin and GTP and FSH and GTP, respectively. The effect on coupling required at least 60 minutes of exposure to TPA or TCDD. Concentration-response curves demonstrated a progressive desensitization with increasing TPA concentration, while TCDD exhibited consistent inhibition over the same concentration range.

  8. Conformational basis for the activation of adenylate cyclase by adenosine

    PubMed Central

    Miles, D. L.; Miles, D. W.; Eyring, H.

    1977-01-01

    The ability of adenosine to stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and increase adenosine 3′:5′-cyclic monophosphate (cAMP) levels has important biochemical consequences. These include the suppression of immune responses and cardiovascular effects. Recent investigations involving the ability of adenosine and adenosine analogs to stimulate adenylate cyclase provided experimental data that appear to be correlated with the ability of adenosine and analogs of adenosine to exist in the glycosidic high anti conformation. 9-β-D-Arabinofuranosyladenine, which is not stable in the high anti conformation, is inactive as a stimulator of adenylate cyclase. 2′-Deoxyadenosine is also not stable in the high anti conformation but its instability may be significantly decreased by intramolecular adjustments promoted by receptor or active site interactions. 2′-Deoxyadenosine does not activate adenylate cyclase in lymphocytes when ATP is the substrate but is able to activate adenylate cyclase when 2-fluoro ATP is the substrate. The inability of certain analogs of adenosine, with bulky groups substituted for hydrogen at the 8 position of the adenine base, to activate adenylate cyclase and increase either lymphocyte or cardiac cell cAMP levels is consistent with the designation of the high anti conformation as being the conformation required for the activation of adenylate cyclase. An understanding of the glycosidic conformation required by the extracellular adenosine receptor of the adenosine molecule provides the basis for designing nucleoside analogs of adenosine that will exert a desired effect on cAMP levels. The avoidance of unwanted immunosuppressive or cardiotoxic effects can be arranged by structural changes that prohibit the high anti conformation. PMID:267918

  9. Adenylate cyclase and carbonic anhydrase in the semicircular canal epithelium of the frog Rana esculenta. An ultrastructural cytochemical localization.

    PubMed

    Oudar, O; Ferrary, E; Feldmann, G

    1990-12-01

    Because the secretion of endolymph has been localized in the ampullar part of the frog semicircular canal, we attempted to determine by cytochemical methods the ultrastructural localization of two enzymes that are assumed to play a role in endolymph secretion: carbonic anhydrase and adenylate cyclase. Functionally, the epithelium of the frog semicircular canal can be schematically divided into three areas: sensory (crista ampullaris), secretory (dark cells), and non-sensory and nonsecretory (transitional and undifferentiated cells) areas. Carbonic anhydrase activity was widely distributed in dark cells. Dark cell labeling disappeared in the presence of acetazolamide. The other cells of the canal did not show any carbonic anhydrase labeling except for the supporting cells of the sensory cells. Adenylate cyclase activity was found on the basolateral and apical membranes of dark cells, and on the apical membrane of sensory cells; weak labeling was also observed in the other epithelial cells. In the apical membrane of the dark cells, adenylate cyclase labeling was dependent on the presence of vasotocin, the frog antidiuretic hormone. The dark cells of the frog semicircular canal thus possess the enzyme equipment needed for the secretion of endolymph and its possible hormonal regulation.

  10. Adenylation by testis-specific cytoplasmic poly(A) polymerase, PAPOLB/TPAP, is essential for spermatogenesis

    PubMed Central

    KASHIWABARA, Shin-ichi; TSURUTA, Satsuki; OKADA, Keitaro; YAMAOKA, Yutaro; BABA, Tadashi

    2016-01-01

    The testis-specific cytoplasmic poly(A) polymerase PAPOLB/TPAP is essential for spermatogenesis. Although this enzyme is responsible for poly(A) tail extension of a subset of mRNAs in round spermatids, the stability and translational efficiency of these mRNAs are unaffected by the absence of PAPOLB. To clarify the functional importance of this enzyme’s adenylation activity, we produced PAPOLB-null mice expressing a polyadenylation-defective PAPOLB mutant (PAPOLBD114A), in which the catalytic Asp at residue 114 was mutated to Ala. Introducing PAPOLBD114A failed to rescue PAPOLB-null phenotypes, such as reduced expression of haploid-specific mRNAs, spermiogenesis arrest, and male infertility. These results suggest that PAPOLB regulates spermatogenesis through its adenylation activity. PMID:27647534

  11. Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments.

    PubMed

    Imbert, M; Chabardès, D; Montégut, M; Clique, A; Morel, F

    1975-01-01

    A method is described, which allows adenylate cyclase activity measurement in single pieces of various nephron segments. Tubular samples of 0.5 to 2 mm length were isolated by microdissection from collagenase treated slices of rabbit kidney. A photograph of each piece was taken in order to measure its length. After a permeabilisation treatment involving preincubation in a hypoosmotic medium and a freezing step, each sample was incubated for 30 mm at 30 degrees C in a medium containing high specific (alpha-32-P)-ATP 3-10-4 M, final volume 2.5 mu 1. The (32P)-cAMP formed was separated from the other labelled nucleotides by filtering the incubate on a dry aluminium oxide microcolumn, 3H cAMP was added as a tracer for measuring cAMP recovery. The sensitivity of the method was found to be a few fentomoles (10-15 M) cAMP. cAMP generation increased linearly as a function of the incubation time up to more than 30 min, and as a function of the length of the segment used. Control and fluoride (5 mM) stimulated adenvlate cyclase activities were measured in the following segments of the nephron: early proximal convoluted tubule (PCT), pars recta of the proximal tubule (PR), thin descending limb of the loop (TDL), cortical portion of the thick ascending limb (CAL), distal convoluted tubule (dct), first branched portion of the collecting tubule (BCT), further cortical (CCT) and medullary (MCT) portions of the collecting tubule. Mean control adenylate cyclase activity varied from 7 (PR) to 75 (BCT) fmoles/mm/30 min. Flouride addition resulted in a 10 (BCT) to 50 (PR) fold increase in enzyme activity. Series of replicates gave a scatter equal to plus or minus 20% (S.D. as a per cent of the mean). The method described appears to be suitable to determine which nephron segments contain hormone-dependent adenylate cyclase.

  12. Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct, Highly Flexible Allosteric Bicarbonate Binding Pocket

    PubMed Central

    Saalau-Bethell, Susanne M; Berdini, Valerio; Cleasby, Anne; Congreve, Miles; Coyle, Joseph E; Lock, Victoria; Murray, Christopher W; O'Brien, M Alistair; Rich, Sharna J; Sambrook, Tracey; Vinkovic, Mladen; Yon, Jeff R; Jhoti, Harren

    2014-01-01

    Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5′-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein. PMID:24616449

  13. Histamine-, norepinephrine-, and dopamine-sensitive central adenylate cyclases: effects of chlorpromazine derivatives and butaclamol.

    PubMed

    Palmer, G C; Wagner, H R; Palmer, S J; Manian, A A

    1978-06-01

    A series of recently available derivatives (quaternary and hydroxylated) of chlorpromazine (CPZ) and butaclamol were evaluated with respect to antagonism of norepinephrine- (NE) (rat cerebral cortex), dopamine- (DA) (rat striatum) and histamine- (H) sensitive (rabbit cerebral cortex) adenylate cyclases. With incubated tissue slices (rat and rabbit cortices) CPZ-CH3, 7-OH-CPZ-CH3, beta-OH-CPZ and butaclamol displayed a capacity to inhibit either NE- or H- induced accumulation of adenosine cyclic 3',5'-monophosphate (cAMP). With the broken cellular enzyme responsive to DA, rather potent inhibition of enzyme activity (IC50 less than 24 micron) occurred with butaclamol, beta-OH-CPZ, 7,8,beta-triOH-CPZ, 7,8-dioxo-beta-OH-CPZ and 3,7,8-triOH-CPZ. It is concluded that the metabolites of CPZ contribute to the central therapeutic and/or side effects of the parent compound.

  14. Coupled ATPase-adenylate kinase activity in ABC transporters.

    PubMed

    Kaur, Hundeep; Lakatos-Karoly, Andrea; Vogel, Ramona; Nöll, Anne; Tampé, Robert; Glaubitz, Clemens

    2016-12-22

    ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on (31)P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters.

  15. Coupled ATPase-adenylate kinase activity in ABC transporters

    PubMed Central

    Kaur, Hundeep; Lakatos-Karoly, Andrea; Vogel, Ramona; Nöll, Anne; Tampé, Robert; Glaubitz, Clemens

    2016-01-01

    ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters. PMID:28004795

  16. Adenylate cyclase in striatal cholinergic interneurons regulates acetylcholine release.

    PubMed

    Login, I S; Hewlett, E L

    1996-10-07

    Fractional [3H]ACH efflux from dissociated rat striata tested whether tonic inhibition prevents stimulation of acetylcholine (ACH) release by adenylate cyclase. Forskolin stimulated release from the dissociated cells (threshold at 300 nM; EC50 > or = 1 MicroM). Release was also stimulated by 3-isobutyl-1-methylxanthine and was additive with forskolin. The 1,9-dideoxy forskolin analog that lacks cyclase-stimulating activity was ineffective. Thus, stimulation of adenylate cyclase within striatal cholinergic interneurons increases ACH secretion but is tonically inhibited by endogenous striatal transmitters. Disinhibition of the excitatory cyclase by denervation of striatal cholinergic interneurons in situ could contribute to supersensitivity without receptor upregulation.

  17. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

    PubMed Central

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F.; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  18. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    PubMed

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

  19. Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate.

    PubMed Central

    Arnez, J G; Harris, D C; Mitschler, A; Rees, B; Francklyn, C S; Moras, D

    1995-01-01

    The crystal structure at 2.6 A of the histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate has been determined. The enzyme is a homodimer with a molecular weight of 94 kDa and belongs to the class II of aminoacyl-tRNA synthetases (aaRS). The asymmetric unit is composed of two homodimers. Each monomer consists of two domains. The N-terminal catalytic core domain contains a six-stranded antiparallel beta-sheet sitting on two alpha-helices, which can be superposed with the catalytic domains of yeast AspRS, and GlyRS and SerRS from Thermus thermophilus with a root-mean-square difference on the C alpha atoms of 1.7-1.9 A. The active sites of all four monomers are occupied by histidyl-adenylate, which apparently forms during crystallization. The 100 residue C-terminal alpha/beta domain resembles half of a beta-barrel, and provides an independent domain oriented to contact the anticodon stem and part of the anticodon loop of tRNA(His). The modular domain organization of histidyl-tRNA synthetase reiterates a repeated theme in aaRS, and its structure should provide insight into the ability of certain aaRS to aminoacylate minihelices and other non-tRNA molecules. Images PMID:7556055

  20. Structure of the adenylation-peptidyl carrier protein didomain of the Microcystis aeruginosa microcystin synthetase McyG.

    PubMed

    Tan, Xiao-Feng; Dai, Ya-Nan; Zhou, Kang; Jiang, Yong-Liang; Ren, Yan-Min; Chen, Yuxing; Zhou, Cong-Zhao

    2015-04-01

    Microcystins, which are the most common cause of hepatotoxicity associated with cyanobacterial water blooms, are assembled in vivo on a large multienzyme complex via a mixed nonribosomal peptide synthetase/polyketide synthetase (NRPS/PKS). The biosynthesis of microcystin in Microcystis aeruginosa PCC 7806 starts with the enzyme McyG, which contains an adenylation-peptidyl carrier protein (A-PCP) didomain for loading the starter unit to assemble the side chain of an Adda residue. However, the catalytic mechanism remains unclear. Here, the 2.45 Å resolution crystal structure of the McyG A-PCP didomain complexed with the catalytic intermediate L-phenylalanyl-adenylate (L-Phe-AMP) is reported. Each asymmetric unit contains two protein molecules, one of which consists of the A-PCP didomain and the other of which comprises only the A domain. Structural analyses suggest that Val227 is likely to be critical for the selection of hydrophobic substrates. Moreover, two distinct interfaces demonstrating variable crosstalk between the PCP domain and the A domain were observed. A catalytic cycle for the adenylation and peptide transfer of the A-PCP didomain is proposed.

  1. [Regularities of organ-specific expression of enzyme systems in cattle].

    PubMed

    Tatarenko, O F; Glazko, V I

    1992-01-01

    The organ specificity of creatine kinase, esterase, isocitrate dehydrogenase lactate dehydrogenase, nucleoside phosphorylase, adenylate kinase, hexokinase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase of black-white cattle has been studied. Esterases, creatine kinase, adenylate kinase, hexokinase and glucose-6-phosphate dehydrogenase have a very wide spectrum of the organ variabilities. Liver and heart have the largest specificity of enzymes activity. Some peculiarities of isozyme spectrum are found in ovaries and spleen.

  2. Crystal structure of aspartyl-tRNA synthetase from Pyrococcus kodakaraensis KOD: archaeon specificity and catalytic mechanism of adenylate formation.

    PubMed Central

    Schmitt, E; Moulinier, L; Fujiwara, S; Imanaka, T; Thierry, J C; Moras, D

    1998-01-01

    The crystal structure of aspartyl-tRNA synthetase (AspRS) from Pyrococcus kodakaraensis was solved at 1.9 A resolution. The sequence and three-dimensional structure of the catalytic domain are highly homologous to those of eukaryotic AspRSs. In contrast, the N-terminal domain, whose function is to bind the tRNA anticodon, is more similar to that of eubacterial enzymes. Its structure explains the unique property of archaeal AspRSs of accommodating both tRNAAsp and tRNAAsn. Soaking the apo-enzyme crystals with ATP and aspartic acid both separately and together allows the adenylate formation to be followed. Due to the asymmetry of the dimeric enzyme in the crystalline state, different steps of the reaction could be visualized within the same crystal. Four different states of the aspartic acid activation reaction could thus be characterized, revealing the functional correlation of the observed conformational changes. The binding of the amino acid substrate induces movement of two invariant loops which secure the position of the peptidyl moiety for adenylate formation. An unambiguous spatial and functional assignment of three magnesium ion cofactors can be made. This study shows the important role of residues present in both archaeal and eukaryotic AspRSs, but absent from the eubacterial enzymes. PMID:9724658

  3. Activation of fat cell adenylate cyclase by protein kinase C

    SciTech Connect

    Naghshineh, S.; Noguchi, M.; Huang, K.P.; Londos, C.

    1986-05-01

    Purified protein kinase C (C-kinase) from guinea pig pancreas and rat brain stimulated adenylate cyclase activity in purified rat adipocyte membranes. Cyclase stimulation occurred over 100 to 1000 mU/ml of C-kinase activity, required greater than 10 ..mu..M calcium, proceeded without a lag, was not readily reversible, and required no exogenous phospholipid. Moreover, C-kinase inhibitors, such as chlorpromazine and palmitoyl carnitine, inhibited selectively adenylate cyclase which was activated by C-kinase and calcium. Depending on assay conditions, 10 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) either enhanced or was required for kinase action on cyclase. Also, TPA plus calcium promoted the quantitative association of C-kinase with membranes. Adenylate cyclase activation by C-kinase was seen both in the presence and absence of exogenous GTP, indicating that the kinase effect does not result from an action on the GTP-binding, inhibitory regulatory component (N/sub i/) of the cyclase system. Moreover, the kinase effect was seen in the presence of non-phosphorylating ATP analogs, such as AppNHp and AppCH/sub 2/p, suggesting that the effects of C-kinase described herein may result from association with, rather than phosphorylation of, adenylate cyclase.

  4. Interaction of 7-bromoacetyl-7-desacetylforskolin with adenylate cyclase

    SciTech Connect

    Laurenza, A.; Morris, D.I.; Seamon, K.B.

    1986-05-01

    7-Bromoacetyl-7-desacetylforskolin (BrAcFk) and the 12-tritio derivative (/sup 3/H-BrAckFk) were synthesized as alkylating analogs of forskolin. BrAcFk stimulated adenylate cyclase in human platelet and bovine brain membranes with an EC50 of 50..mu..M and inhibited /sup 3/H-forskolin binding to these membranes with a K/sub i/ of 300 nM. /sup 3/H-forskolin binding was decreased in membranes pretreated for 20 min with 10 ..mu..M BrAcFk. The i,9-dideoxy derivative of BrAcFk did not activate adenylate cyclase or inhibit /sup 3/H-forskolin binding. Proteins labelled by BrAcFk in solubilized preparations from bovine brain and human platelets were identified by fluorography of SDS gels. The two predominant bands labelled in the low and high molecular weight regions had molecular weights of 50,000 and 135,000 daltons respectively. The 135,000 dalton band identified by fluorography coeluted with adenylate cyclase activity on a Dupont GF450 column and has a molecular weight identical to that of the catalytic subunit determined by silver staining of SDS gels. These results suggest that BrAcFk can react covalently with the catalytic subunit of adenylate cyclase.

  5. Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin.

    PubMed Central

    Gueirard, P; Guiso, N

    1993-01-01

    Bordetella bronchiseptica is a pathogen of laboratory, domestic, and wild animals and sometimes of humans. In the present study some characteristics of the virulence of B. bronchiseptica isolates of different origin were studied. All isolates had similar phenotypes, similar bacteriological characters, and synthesized adenylate cyclase-hemolysin, filamentous hemagglutinin and pertactin but not pertussis toxin. These isolates, however, differed in their ability to express dermonecrotic toxin and to cause a lethal infection, but no correlation was found with the human or animal origin of the isolates. The fact that the most virulent isolate did not express dermonecrotic toxin suggests that this toxin does not play an important role in the virulence of the bacteria in the murine model. After infection with virulent B. bronchiseptica a very early synthesis and a persistence of anti-adenylate cyclase-hemolysin and anti-filamentous hemagglutinin antibodies were observed in the sera of infected mice, suggesting a persistence of the bacteria or of its antigens. B. bronchiseptica adenylate cyclase-hemolysin was purified and was shown to be a major protective antigen against B. bronchiseptica infection. Furthermore, we showed that its immunological and protective properties were different from that of B. pertussis adenylate cyclase-hemolysin, confirming that Bordetella species are immunologically different. Images PMID:8406794

  6. Irreversible stimulation of adenylate cyclase activity of fat cell membranes of phosphoramidate and phosphonate analogs of GTP.

    PubMed

    Cuatrecasas, P; Bennett, V; Jacobs, S

    1975-01-01

    The ability of 5'-guanylylimidodiphosphate (Gpp(NH)p) to stimulate irreversibly the adenylate cyclease activity of fat cell membranes has been studied by preincubating the membranes with this or related analogs followed by assaying after thoroughly washing the membranes. Activation can occur in a simple Tris-HCl buffer, in the absence of added divalent cations and in the presence of EDTA. Dithiothreitol enhances the apparent degree of activation, perhaps by stabilization. The importance of utilizing optimal conditions for stabilizing enzyme activity, and of measuring the simultaneous changes in the control enzyme, is illustrated. The organomercurial, p-aminophenylmercuric acetate, inhibits profoundly the activity of the native as well as the Gpp(NH)p-stimulated adenylate cyclase, but in both cases subsequent exposure to dithiothreitol restores fully the original enzyme activity. However, the mercurial-inactivated enzyme does not react with Gpp(NP)p, as evidenced by the subsequent restoration of only the control enzyme activity upon exposure to dithiothreitol. Thus, reaction with Gpp(NH)p requires intact sulfhydryl groups, but the activated state is not irreversibly destroyed by the inactivation caused by sulfhydryl blockade. GTP and, less effectively, GDP and ATP inhibit activation by Gpp(NH)p, but interpretations are complicated by the facts that this inhibition is overcome with time and that GTP and ATP can protect potently from spontaneous inactivation. These two nucleotides can be used in the Gpp(NH)p preincubation to stabilize the enzyme. The Gpp(NH)p-activated enzyme cannot be reversed spontaneously during prolonged incubation at 30 degrees C in the absence or presence of GTP, ATP, MgCl2, glycine, dithiothreitol, NaF or EDTA. The strong nucleophile, neutral hydroxylamine, decreases the Gpp(NH)p-activated enzyme activity and no subsequent activation is detected upon re-exposure to the nucleotide.

  7. Structure of the adenylation domain Thr1 involved in the biosynthesis of 4-chlorothreonine in Streptomyces sp. OH-5093-protein flexibility and molecular bases of substrate specificity.

    PubMed

    Scaglione, Antonella; Fullone, Maria Rosaria; Montemiglio, Linda Celeste; Parisi, Giacomo; Zamparelli, Carlotta; Vallone, Beatrice; Savino, Carmelinda; Grgurina, Ingeborg

    2017-09-01

    We determined the crystal structure of Thr1, the self-standing adenylation domain involved in the nonribosomal-like biosynthesis of free 4-chlorothreonine in Streptomyces sp. OH-5093. Thr1 shows two monomers in the crystallographic asymmetric unit with different relative orientations of the C- and N-terminal subdomains both in the presence of substrates and in the unliganded form. Cocrystallization with substrates, adenosine 5'-triphosphate and l-threonine, yielded one monomer containing the two substrates and the other in complex with l-threonine adenylate, locked in a postadenylation state. Steady-state kinetics showed that Thr1 activates l-Thr and its stereoisomers, as well as d-Ala, l- and d-Ser, albeit with lower efficiency. Modeling of these substrates in the active site highlighted the molecular bases of substrate discrimination. This work provides the first crystal structure of a threonine-activating adenylation enzyme, a contribution to the studies on conformational rearrangement in adenylation domains and on substrate recognition in nonribosomal biosynthesis. Structural data are available in the Protein Data Bank under the accession number 5N9W and 5N9X. © 2017 Federation of European Biochemical Societies.

  8. Nucleotide-gated KATP channels integrated with creatine and adenylate kinases: Amplification, tuning and sensing of energetic signals in the compartmentalized cellular environment

    PubMed Central

    Selivanov, Vitaliy A.; Alekseev, Alexey E.; Hodgson, Denice M.; Dzeja, Petras P.; Terzic, Andre

    2009-01-01

    Transmission of energetic signals to membrane sensors, such as the ATP-sensitive K+(KATP) channel, is vital for cellular adaptation to stress. Yet, cell compartmentation implies diffusional hindrances that hamper direct reception of cytosolic energetic signals. With high intracellular ATP levels, KATP channels may sense not bulk cytosolic, but rather local submembrane nucleotide concentrations set by membrane ATPases and phosphotransfer enzymes. Here, we analyzed the role of adenylate kinase and creatine kinase phosphotransfer reactions in energetic signal transmission over the strong diffusional barrier in the submembrane compartment, and translation of such signals into a nucleotide response detectable by KATP channels. Facilitated diffusion provided by creatine kinase and adenylate kinase phosphotransfer dissipated nucleotide gradients imposed by membrane ATPases, and shunted diffusional restrictions. Energetic signals, simulated as deviation of bulk ATP from its basal level, were amplified into an augmented nucleotide response in the submembrane space due to failure under stress of creatine kinase to facilitate nucleotide diffusion. Tuning of creatine kinase-dependent amplification of the nucleotide response was provided by adenylate kinase capable of adjusting the ATP/ADP ratio in the submembrane compartment securing adequate KATP channel response in accord with cellular metabolic demand. Thus, complementation between creatine kinase and adenylate kinase systems, here predicted by modeling and further supported experimentally, provides a mechanistic basis for metabolic sensor function governed by alterations in intracellular phosphotransfer fluxes. PMID:14977185

  9. Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes.

    PubMed

    Novak, Jakub; Cerny, Ondrej; Osickova, Adriana; Linhartova, Irena; Masin, Jiri; Bumba, Ladislav; Sebo, Peter; Osicka, Radim

    2017-09-24

    Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (αMβ₂) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA toxin action, placing particular emphasis on signaling mechanisms by which the toxin-produced 3',5'-cyclic adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.

  10. Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

    SciTech Connect

    Slotkin, T.A.; Navarro, H.A.; McCook, E.C.; Seidler, F.J. )

    1990-01-01

    Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a {beta}-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of {beta}-adrenergic receptors: in fact, ({sup 125}I)pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.

  11. Effect of hypolipidemic drugs on basal and stimulated adenylate cyclase activity in tumor cells

    SciTech Connect

    Bershtein, L.M.; Kovaleva, I.G.; Rozenberg, O.A.

    1986-02-01

    This paper studies adenylate cyclase acticvity in Ehrlich's ascites carcinoma (EAC) cells during administration of drugs with a hypolipidemic action. Seven to eight days before they were killed, male mice ingested the antidiabetic biguanide phenformin, and the phospholipid-containing preparation Essentiale in drinking water. The cAMP formed was isolated by chromatography on Silufol plates after incubation of the enzyme preparation with tritium-ATP, or was determined by the competitive binding method with protein. It is shown that despite the possible differences in the concrete mechanism of action of the hypolipidemic agents chosen for study on the cyclase system, the use of such agents, offers definite prospects for oriented modification of the hormone sensitivity of tumor cells.

  12. Aminoacyl transfer from an adenylate anhydride to polyribonucleotides

    NASA Technical Reports Server (NTRS)

    Weber, A. L.; Lacey, J. C., Jr.

    1975-01-01

    Imidazole catalysis of phenylalanyl transfer from phenylalanine adenylate to hydroxyl groups of homopolyribonucleotides is studied as a possible chemical model of biochemical aminoacylation of transfer RNA (tRNA). The effect of pH on imidazole-catalyzed transfer of phenylalanyl residues to poly(U) and poly(A) double helix strands, the number of peptide linkages and their lability to base and neutral hydroxylamine, and the nature of adenylate condensation products are investigated. The chemical model entertained exhibits a constraint by not acylating the hydroxyl groups of polyribonucleotides in a double helix. The constraint is consistent with selective biochemical aminoacylation at the tRNA terminus. Interest in imidazole as a model of histidine residue in protoenzymes participating in prebiotic aminoacyl transfer to polyribonucleotides, and in rendering the tRNA a more efficient adaptor, is indicated.

  13. Interaction of Trypanosoma cruzi adenylate cyclase with liver regulatory factors.

    PubMed Central

    Eisenschlos, C; Flawiá, M M; Torruella, M; Torres, H N

    1986-01-01

    Trypanosoma cruzi adenylate cyclase catalytic subunits may interact with regulatory factors from rat liver membranes, reconstituting heterologous systems which are catalytically active in assay mixtures containing MgATP. The systems show stimulatory responses to glucagon and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) or fluoride. Reconstitution was obtained by three different methods: fusion of rat liver membranes (pretreated with N-ethylmaleimide) to T. cruzi membranes; interaction of detergent extracts of rat liver membranes with T. cruzi membranes; or interaction of purified preparations of T. cruzi adenylate cyclase and of liver membrane factors in phospholipid vesicles. The liver factors responsible for the guanine nucleotide effect were characterized as the NS protein. Data also indicate that reconstitution requires the presence of a membrane substrate. PMID:2947568

  14. Aminoacyl transfer from an adenylate anhydride to polyribonucleotides

    NASA Technical Reports Server (NTRS)

    Weber, A. L.; Lacey, J. C., Jr.

    1975-01-01

    Imidazole catalysis of phenylalanyl transfer from phenylalanine adenylate to hydroxyl groups of homopolyribonucleotides is studied as a possible chemical model of biochemical aminoacylation of transfer RNA (tRNA). The effect of pH on imidazole-catalyzed transfer of phenylalanyl residues to poly(U) and poly(A) double helix strands, the number of peptide linkages and their lability to base and neutral hydroxylamine, and the nature of adenylate condensation products are investigated. The chemical model entertained exhibits a constraint by not acylating the hydroxyl groups of polyribonucleotides in a double helix. The constraint is consistent with selective biochemical aminoacylation at the tRNA terminus. Interest in imidazole as a model of histidine residue in protoenzymes participating in prebiotic aminoacyl transfer to polyribonucleotides, and in rendering the tRNA a more efficient adaptor, is indicated.

  15. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    SciTech Connect

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of ({sup 3}H)GDP binding to plasma membranes suggested a single high affinity site with a K{sub d} = 0.24 uM. Competition studies indicated that GTP{sub {gamma}}S was 7-fold more potent than GDP{sub {beta}}S. Bound GDP could be released by FSH in the presence of GTP{sub {gamma}}S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP{sub {beta}}S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP{sub {beta}}S competitively inhibited GTP{sub {gamma}}S-stimulated adenylate cyclase activity with a K{sub i} = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP{sub {gamma}}S-bound form persisted even if GDP{sub {beta}}S previously occupied all available binding sites. Two membrane proteins, M{sub r} = 43,000 and 48,000, were ADP{centered dot}ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP{sub {gamma}}S but not by GDP{sub {beta}}S. The M{sub r} = 43,000 and 48,000 proteins represented variant forms of G{sub S}. A single protein of M{sub r} = 40,000 (G{sub i}) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC{sub 50} = 0.1 uM. The adenosine analog, N{sup 6}{centered dot}phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin.

  16. [Adenylate cyclase. A possible factor in the pathogenicity of Yersinia pestis].

    PubMed

    Michankin, B N; Chevchenko, L A; Asseeva, L E

    1992-01-01

    Biological effect of homogenous preparation of Y. pestis adenylate cyclase on eucaryotic cells was studied. Adenylate cyclase, added (7.5 x 10(8) g/ml) to guinea pig macrophages lowers the level of chemiluminescence to 50-70%, has an appreciable cytotoxic effect on peritoneal macrophages and suppresses phosphorylation processes of leucocyte proteins from white mice. The experimental results obtained allow to suggest Y. pestis adenylate cyclase to be a pathogenic factor, contributing to the development of plague infection.

  17. Structure and function of adenylate kinase isozymes in normal humans and muscular dystrophy patients.

    PubMed

    Hamada, M; Takenaka, H; Fukumoto, K; Fukamachi, S; Yamaguchi, T; Sumida, M; Shiosaka, T; Kurokawa, Y; Okuda, H; Kuby, S A

    1987-01-01

    Two isozymes of adenylate kinase from human Duchenne muscular dystrophy serum, one of which was an aberrant form specific to DMD patients, were separated by Blue Sepharose CL-6B affinity chromatography. The separated aberrant form possessed a molecular weight of 98,000 +/- 1,500, whereas the normal serum isozyme had a weight of 87,000 +/- 1,600, as determined by SDS-polyacrylamide gel electrophoresis, gel filtration, and sedimentation equilibrium. The sedimentation coefficients were 5.8 S and 5.6 S for the aberrant form and the normal form, respectively. Both serum isozymes are tetramers. The subunit size of the aberrant isozyme (Mr = 24,700) was very similar to that of the normal human liver isozyme, and the subunit size of the normal isozyme (Mr = 21,700) was very similar to that of the normal human muscle enzyme. The amino acid composition of the normal serum isozyme was similar to that of the muscle-type enzyme, and that of the aberrant isozyme was similar to that of the liver enzyme, with some exceptions in both cases.

  18. Signal-transduction protein P(II) from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.

    PubMed

    Fokina, Oleksandra; Herrmann, Christina; Forchhammer, Karl

    2011-11-15

    P(II) proteins belong to a family of highly conserved signal-transduction proteins that occurs widely in bacteria, archaea and plants. They respond to the central metabolites ATP, ADP and 2-OG (2-oxoglutarate), and control enzymes, transcription factors and transport proteins involved in nitrogen metabolism. In the present study, we examined the effect of ADP on in vitro P(II)-signalling properties for the cyanobacterium Synechococcus elongatus, a model for oxygenic phototrophic organisms. Different ADP/ATP ratios strongly affected the properties of P(II) signalling. Increasing ADP antagonized the binding of 2-OG and directly affected the interactions of P(II) with its target proteins. The resulting P(II)-signalling properties indicate that, in mixtures of ADP and ATP, P(II) trimers are occupied by mixtures of adenylate nucleotides. Binding and kinetic activation of NAGK (N-acetyl-L-glutamate kinase), the controlling enzyme of arginine biosynthesis, by P(II) was weakened by ADP, but relief from arginine inhibition remained unaffected. On the other hand, ADP enhanced the binding of P(II) to PipX, a co-activator of the transcription factor NtcA and, furthermore, antagonized the inhibitory effect of 2-OG on P(II)-PipX interaction. These results indicate that S. elongatus P(II) directly senses the adenylate energy charge, resulting in target-dependent differential modification of the P(II)-signalling properties.

  19. Minimum Free Energy Path of Ligand-Induced Transition in Adenylate Kinase

    PubMed Central

    Matsunaga, Yasuhiro; Fujisaki, Hiroshi; Terada, Tohru; Furuta, Tadaomi; Moritsugu, Kei; Kidera, Akinori

    2012-01-01

    Large-scale conformational changes in proteins involve barrier-crossing transitions on the complex free energy surfaces of high-dimensional space. Such rare events cannot be efficiently captured by conventional molecular dynamics simulations. Here we show that, by combining the on-the-fly string method and the multi-state Bennett acceptance ratio (MBAR) method, the free energy profile of a conformational transition pathway in Escherichia coli adenylate kinase can be characterized in a high-dimensional space. The minimum free energy paths of the conformational transitions in adenylate kinase were explored by the on-the-fly string method in 20-dimensional space spanned by the 20 largest-amplitude principal modes, and the free energy and various kinds of average physical quantities along the pathways were successfully evaluated by the MBAR method. The influence of ligand binding on the pathways was characterized in terms of rigid-body motions of the lid-shaped ATP-binding domain (LID) and the AMP-binding (AMPbd) domains. It was found that the LID domain was able to partially close without the ligand, while the closure of the AMPbd domain required the ligand binding. The transition state ensemble of the ligand bound form was identified as those structures characterized by highly specific binding of the ligand to the AMPbd domain, and was validated by unrestrained MD simulations. It was also found that complete closure of the LID domain required the dehydration of solvents around the P-loop. These findings suggest that the interplay of the two different types of domain motion is an essential feature in the conformational transition of the enzyme. PMID:22685395

  20. Minimum free energy path of ligand-induced transition in adenylate kinase.

    PubMed

    Matsunaga, Yasuhiro; Fujisaki, Hiroshi; Terada, Tohru; Furuta, Tadaomi; Moritsugu, Kei; Kidera, Akinori

    2012-01-01

    Large-scale conformational changes in proteins involve barrier-crossing transitions on the complex free energy surfaces of high-dimensional space. Such rare events cannot be efficiently captured by conventional molecular dynamics simulations. Here we show that, by combining the on-the-fly string method and the multi-state Bennett acceptance ratio (MBAR) method, the free energy profile of a conformational transition pathway in Escherichia coli adenylate kinase can be characterized in a high-dimensional space. The minimum free energy paths of the conformational transitions in adenylate kinase were explored by the on-the-fly string method in 20-dimensional space spanned by the 20 largest-amplitude principal modes, and the free energy and various kinds of average physical quantities along the pathways were successfully evaluated by the MBAR method. The influence of ligand binding on the pathways was characterized in terms of rigid-body motions of the lid-shaped ATP-binding domain (LID) and the AMP-binding (AMPbd) domains. It was found that the LID domain was able to partially close without the ligand, while the closure of the AMPbd domain required the ligand binding. The transition state ensemble of the ligand bound form was identified as those structures characterized by highly specific binding of the ligand to the AMPbd domain, and was validated by unrestrained MD simulations. It was also found that complete closure of the LID domain required the dehydration of solvents around the P-loop. These findings suggest that the interplay of the two different types of domain motion is an essential feature in the conformational transition of the enzyme.

  1. Forskolin inhibits the Gs-stimulated adenylate cyclase in rat ascites hepatoma AH66F cells.

    PubMed

    Miyamoto, K; Sanae, F; Koshiura, R; Matsunaga, T; Hasegawa, T; Takagi, K; Satake, T

    1989-09-01

    Forskolin increased intracellular cyclic AMP and augmented cyclic AMP formation by prostaglandin E1 (PGE1) in normal rat hepatocytes and ascites hepatoma AH66 cells. However, in AH66F cells which were derived from the AH66 cell line, the diterpene only slightly increased the cyclic AMP level, and dose-dependently inhibited the accumulation caused by PGE1. Forskolin dose-dependently activated adenylate cyclase in these membranes, and the magnitude of activation by forskolin was largest in the following order: hepatocytes, AH66 cells, and AH66F cells. This difference may be based on the number of forskolin-binding sites. The binding affinity of forskolin for each cell membrane was similar. The number and affinity of forskolin-binding sites in these cells were not influenced by 5'-guanylylimidodiphosphate [Gpp(NH)p]. In hepatocytes and AH66 cells, forskolin and other adenylate cyclase activators such as PGE1, GTP, Gpp(NH)p, F-, and Mn2+ synergistically increased the enzyme activity. In AH66F cells, the forskolin-stimulated activity was hardly influenced by the GTP analog, and forskolin diminished the activities induced by the GTP analog in a manner similar to that of diterpene alone. Forskolin (10 microM) also significantly inhibited the activities induced by PGE1, GTP, and F-. The effect of forskolin with Mn2+ was additive in AH66F cells. The data suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide-binding protein and the catalytic unit in the membrane of normal hepatocytes and AH66 cells, but it interferes with the coupling in AH66F cells.

  2. Design and Synthesis of Fluorescent Acyclic Nucleoside Phosphonates as Potent Inhibitors of Bacterial Adenylate Cyclases.

    PubMed

    Břehová, Petra; Šmídková, Markéta; Skácel, Jan; Dračínský, Martin; Mertlíková-Kaiserová, Helena; Velasquez, Monica P Soto; Watts, Val J; Janeba, Zlatko

    2016-11-21

    Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF) are key virulence factors with adenylate cyclase (AC) activity that substantially contribute to the pathogenesis of whooping cough and anthrax, respectively. There is an urgent need to develop potent and selective inhibitors of bacterial ACs with prospects for the development of potential antibacterial therapeutics and to study their molecular interactions with the target enzymes. Novel fluorescent 5-chloroanthraniloyl-substituted acyclic nucleoside phosphonates (Cl-ANT-ANPs) were designed and synthesized in the form of their diphosphates (Cl-ANT-ANPpp) as competitive ACT and EF inhibitors with sub-micromolar potency (IC50 values: 11-622 nm). Fluorescence experiments indicated that Cl-ANT-ANPpp analogues bind to the ACT active site, and docking studies suggested that the Cl-ANT group interacts with Phe306 and Leu60. Interestingly, the increase in direct fluorescence with Cl-ANT-ANPpp having an ester linker was strictly calmodulin (CaM)-dependent, whereas Cl-ANT-ANPpp analogues with an amide linker, upon binding to ACT, increased the fluorescence even in the absence of CaM. Such a dependence of binding on structural modification could be exploited in the future design of potent inhibitors of bacterial ACs. Furthermore, one Cl-ANT-ANP in the form of a bisamidate prodrug was able to inhibit B. pertussis ACT activity in macrophage cells with IC50 =12 μm. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Restoration of adenylate cyclase responsiveness in murine myeloid leukemia permits inhibition of proliferation by hormone. Butyrate augments catalytic activity of adenylate cyclase.

    PubMed

    Inhorn, L; Fleming, J W; Klingberg, D; Gabig, T G; Boswell, H S

    1988-04-01

    Mechanisms of leukemic cell clonal dominance may include aberrations of transmembrane signaling. In particular, neoplastic transformation has been associated with reduced capacity for hormone-stimulated adenylate cyclase activity. In the present study, prostaglandin E, a hormonal activator of adenylate cyclase that has antiproliferative activity in myeloid cells, and cholera toxin, an adenylate cyclase agonist that functions at a postreceptor site by activating the adenylate cyclase stimulatory GTP-binding protein (Gs), were studied for antiproliferative activity in two murine myeloid cell lines. FDC-P1, an interleukin 3 (IL 3)-dependent myeloid cell line and a tumorigenic IL 3-independent subline, FI, were resistant to these antiproliferative agents. The in vitro ability of the "differentiation" agent, sodium butyrate, to reverse their resistance to adenylate cyclase agonists was studied. The antiproliferative action of butyrate involved augmentation of transmembrane adenylate cyclase activity. Increased adenylate cyclase catalyst activity was the primary alteration of this transmembrane signaling group leading to the functional inhibitory effects on leukemia cells, although alterations in regulatory G-proteins appear to play a secondary role.

  4. Desensitization of adenylate cyclase in a human keratinocyte cell line by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

    SciTech Connect

    Choi, E.J.; Young, M.J.; Toscano, D.L.; Greenlee, W.F.; Toscano, W.A. Jr.

    1987-05-01

    Regulation of adenylate cyclase in human keratinocyte cell line SCC 12 is altered after TCDD exposure. TCDD-treated cells show a 50% decrease in isoproterenol - stimulated adenylate cyclase activity. The reduced responsiveness of these cells to isoproterenol was concentration dependent on TCDD. The inactive TCDD analog, 2,7-dibenzo-p-dioxin did not affect isoproterenol activation. Altered hormone stimulation of adenylate cyclase can result from decreased receptor number or affinity, a defect in coupling of receptors via G/sub s/, or modification of the catalytic subunit. To distinguish between these possibilities, enzyme activity was assayed in the presence of different site-specific activators of this enzyme system. Cells exposed to TCDD for 24 hr showed a reduced response to the GTP analog, Gpp(NH)p. Forskolin stimulation was not affected by TCDD treatment. (/sup 125/I)-iodocyanopindolol (ICP) binding to ..beta..-adrenergic receptors was examined after TCDD treatment. The equilibrium dissociation constant (K/sub d/) for ICP was unaffected by TCDD treatment, whereas, the total number of specific ICP-binding sites was reduced from 1080 in control cells to 780 sites per cell in TCDD (10 nM) exposed cells.

  5. (/sup 3/H)forskolin- and (/sup 3/H)dihydroalprenolol-binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    SciTech Connect

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1988-03-01

    The characteristics of the cardiac adenylate cyclase system were studied in rats fed diets containing fish oil (menhaden oil) and other oils. Adenylate cyclase activity generally was higher in cardiac homogenates and membranes of rats fed diet containing 10% menhaden oil than in the other oils. The increase in enzyme activity, especially in forskolin-stimulated activity, was associated with an increase in the concentration of the (/sup 3/H) forskolin-binding sites in cardiac membranes of rats fed menhaden oil. The beta-adrenergic receptor concentration was not significantly altered although the affinity for (/sup 3/H)dihydroalprenolol-binding was lower in membranes of rats fed menhaden oil than those fed the other oils. omega-3 fatty acids from menhaden oil were incorporated into the cardiac membrane phospholipids. The results suggest that the observed increase in myocardial adenylate cyclase activity of rats fed menhaden oil may be due to an increase in the number of the catalytic subunits of the enzyme or due to a greater availability of the forskolin-binding sites.

  6. The invasive adenylate cyclase of Bordetella pertussis. Intracellular localization and kinetics of penetration into various cells.

    PubMed Central

    Farfel, Z; Friedman, E; Hanski, E

    1987-01-01

    The penetration of Bordetella pertussis adenylate cyclase into various mammalian cells exhibits similar kinetics; the accumulation of both intracellular cyclase activity and cyclic AMP is rapid, reaching constant levels after 15-60 min of incubation. The kinetics of enzyme penetration into turkey erythrocytes is different; cyclase activity and cyclic AMP accumulate linearly and do not reach constant levels even after 6 h of incubation. In the preceding paper [Friedman, Farfel & Hanski (1987) Biochem. J. 243, 145-151] we have suggested that the constant level of intracellular cyclase activity reflects a steady state formed by continuous penetration and intracellular inactivation of the enzyme. In contrast with other mammalian cells, no inactivation of cyclase is observed in turkey erythrocytes. These results further support the notion that there is continuous penetration and deactivation of the invasive enzyme in mammalian cells. A 5-6-fold increase in specific activity of the invasive cyclase is detected in a pellet fraction of human lymphocytes in which a similar increase in specific activity of the plasma-membrane marker 5'-nucleotidase is observed. A similar increase in the invasive-cyclase specific activity is detected in a membrane fraction of human erythrocytes. Cyclase activity in a membrane-enriched fraction of human lymphocytes reached a constant level after 20 min of cell exposure to the enzyme. Similar time courses were observed for accumulation of cyclase activity and cyclic AMP in whole lymphocytes [Friedman, Farfel & Hanski (1987) Biochem, J. 243, 145-151]. We suggest therefore that cyclic AMP generation by the invasive enzyme as well as the intracellular inactivation process occur while it is associated with a membrane fraction identical, or closely associated, with the plasma membrane. PMID:2886120

  7. Adenylate energy pool and energy charge in maturing rape seeds.

    PubMed

    Ching, T M; Crane, J M

    1974-11-01

    A study of energy state and chemical composition of pod walls and seeds of maturing rape (Brassica napus L.) was conducted on two varieties, Victor and Gorczanski. Total adenosine phosphates, ATP, and adenylate energy charge increased with increasing cell number and cellular synthesis during the early stages, remained high at maximum dry weight accumulation and maximum substrate influx time, and decreased with ripening. A temporal control of energy supply and ATP concentration is evident in developing tissues with determined functions; whereas the association of a high energy charge and active cellular biosynthesis occurs only in tissues with a stabilized cell number.

  8. Conformational Dynamics of Adenylate Kinase: The Effects of Temperature and Mutation on Friction, Memory, and Reactivity

    NASA Astrophysics Data System (ADS)

    Watkins, Lucas; Duderstadt, Karl; Bhattacharyya, Sucharita; Yang, Haw

    2006-03-01

    Enzymes reside on a convoluted free energy surface. This free energy surface generates the conformational dynamics that control activity. We use single molecule Förster Resonance Energy Transfer measurements to study these conformational dynamics and the physics that underly them in a model enzyme, Adenylate Kinase (AK), which catalyzes the disproportionation of ADP into AMP and ATP. Our microscope records time-dependent single-molecule trajectories as a list of single photon arrival times. We treat the distance trajectory that generates this data as a manifestation of a many-dimensional Langevin equation, projected onto the coordinate defined by our two labeling sites. Using a likelihood-based approach, we can then directly extract the potential of mean force and the friction coefficient from the raw photon-by-photon trajectories. Temperature-dependent studies allow calculation of entropy and enthalpy profiles from the measured potentials of mean force, while mutants in functionally-important regions allow us to understand the role of individual residues in dynamics and catalysis. Ultimately, this newly-developed method allows us to begin to draw direct connections between structure, dynamics, and reactivity.

  9. Molecular mechanism of photoactivation of a light-regulated adenylate cyclase.

    PubMed

    Ohki, Mio; Sato-Tomita, Ayana; Matsunaga, Shigeru; Iseki, Mineo; Tame, Jeremy R H; Shibayama, Naoya; Park, Sam-Yong

    2017-08-08

    The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.

  10. Fluorescence and NMR investigations in the ligand binding properties of adenylate kinases

    SciTech Connect

    Reinstein, J.; Vetter, I.R.; Schlichting, I.; Roesch, P.; Wittinghofer, A.; Goody, R.S. )

    1990-08-14

    A new system for measurement of affinities of adenylate kinases (AK) for substrates and inhibitors is presented. This system is based on the use of the fluorescent ligand {alpha},{omega}-di((3{prime} or 2{prime})-O-(N-methyl-anthraniloyl)adenosine-5{prime}) pentaphosphate (MAP5Am), which is an analogue of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A). It allows the determination of dissociation constants for any ligand in the range of 1 {times} 10{sup {minus}9} to 5 {times} 10{sup {minus}2} M. Affinities for different bisubstrate inhibitors (AP4A, AP5A, AP6A) and substrates (AMP, ADP, ATP, GTP) were determined in the presence and absence of magnesium. An analysis of the binding of bisubstrate inhibitors is proposed and applied to these data. Temperature denaturation experiments indicate that the mutant enzyme has the same thermal stability as the wild-type enzyme and, as NMR studies indicate, also a very similar structure. Together with the results obtained by Tian et al on the effect of replacement of the conserved His-36 in the cytosolic AK (AK1) from chicken by glutamine and asparagine, this shows that residues 28 of AK from E. coli (AKec) and 36 of AK1 are situated in a comparable environment and are not essential for catalytic activity.

  11. Role of adenosine 3',5'-monophosphate and the Ri-receptor Gi-coupled adenylate cyclase inhibitory pathway in the mechanism whereby adrenalectomy increases the adenosine antilipolytic effect in rat fat cells.

    PubMed

    de Mazancourt, P; Lacasa, D; Giot, J; Giudicelli, Y

    1989-03-01

    The aim of this study was to establish the mechanism by which adrenalectomy promotes the antilipolytic effect of the adenosine analog (-)-N6-(R-phenyl-isopropyl)adenosine (R-PIA) in rat fat cells. This action of adrenalectomy was not specific for R-PIA, since it was also observed with nicotinic acid and was prevented by phosphodiesterase inhibitors. In contrast, the inhibitory effect of R-PIA and nicotinic acid toward isoproterenol-stimulated cAMP accumulation was unaltered by adrenalectomy regardless of whether phosphodiesterase inhibitors were present. Whatever the conditions used, however, the cAMP levels in adrenalectomized rat adipocytes were one quarter to one third of those in sham-operated rats and remained below the limit over which variations in cAMP had no more influence in lipolysis. Both total and particulate low Km cAMP phosphodiesterase activities per adipocyte were decreased in adrenalectomized rats, but the stimulatory responses of the particulate enzyme to R-PIA remained unchanged. Pertussis toxin-catalyzed ADP ribosylation studies revealed a marked decrease in the total amount of the alpha-subunits of Go and the adenylate cyclase inhibitory regulatory protein Gi after adrenalectomy. However, the inhibitory dose-response curves of adenylate cyclase to R-PIA, nicotinic acid, GTP, guanylylimidodiphosphate, and guanosine 5'-O-(3-thiotriphosphate) were unaltered by adrenalectomy, indicating that the inhibitory function of Gi is unimpaired by adrenalectomy. Lastly, adrenalectomy resulted in a 60% reduction of the Mn2+-stimulated adenylate cyclase activity/adipocyte, which indicates that adrenalectomy causes a defect in adenylate cyclase catalytic activity. Thus, enhanced antilipolytic effects of R-PIA induced by adrenalectomy do not involve increased function of the adenosine receptor Gi-coupled adenylate cyclase inhibitory pathway, but are related to abnormally low intracellular cAMP levels due to defective adenylate cyclase catalytic activity.

  12. Allosteric equilibrium model explains steady-state coupling of beta-adrenergic receptors to adenylate cyclase in turkey erythrocyte membranes.

    PubMed Central

    Ugur, O; Onaran, H O

    1997-01-01

    We used a simple experimental approach to clarify some contradictory predictions of the collision coupling and equilibrium models (e.g. ternary complex, two-state ternary complex or quinternary complex), which describe G-protein-mediated beta-adrenergic receptor signalling in essentially different manners. Analysis of the steady-state coupling of beta-adrenoceptors to adenylate cyclase in turkey erythrocyte membranes showed that: (1) in the absence of an agonist, Gpp(NH)p (a hydrolysis-resistant analogue of GTP) can activate adenylate cyclase very slowly; (2) this activity reaches a steady state in approx. 5 h, the extent of activity depending on the concentration of the nucleotide; (3) isoprenaline-activated steady-state adenylate cyclase can be inactivated by propranolol (a competitive antagonist that relaxes the receptor activation), in the presence of Gpp(NH)p (which provides a virtual absence of GTPase) and millimolar concentrations of Mg2+ (the rate of this inactivation is relatively fast); (4) increasing the concentration of Gpp(NH)p can saturate the steady-state activity of adenylate cyclase. The saturated enzyme activity was lower than that induced by isoprenaline under the same conditions. This additional agonist-induced activation was reversible. In the light of these results, we conclude that agonist can also activate the guanine nucleotide-saturated system in the absence of GTPase by a mechanism other than guanine nucleotide exchange. We explain these phenomena in the framework of a quinternary complex model as an agonist-induced and receptor-mediated dissociation of guanine nucleotide-saturated residual heterotrimer, the equilibrium concentration of which is not necessarily zero. These results, which suggest a continuous interaction between receptor and G-protein, can hardly be accommodated by the collision coupling model that was originally suggested for the present experimental system and then applied to many other G-protein systems. Therefore we

  13. Allosteric equilibrium model explains steady-state coupling of beta-adrenergic receptors to adenylate cyclase in turkey erythrocyte membranes.

    PubMed

    Ugur, O; Onaran, H O

    1997-05-01

    We used a simple experimental approach to clarify some contradictory predictions of the collision coupling and equilibrium models (e.g. ternary complex, two-state ternary complex or quinternary complex), which describe G-protein-mediated beta-adrenergic receptor signalling in essentially different manners. Analysis of the steady-state coupling of beta-adrenoceptors to adenylate cyclase in turkey erythrocyte membranes showed that: (1) in the absence of an agonist, Gpp(NH)p (a hydrolysis-resistant analogue of GTP) can activate adenylate cyclase very slowly; (2) this activity reaches a steady state in approx. 5 h, the extent of activity depending on the concentration of the nucleotide; (3) isoprenaline-activated steady-state adenylate cyclase can be inactivated by propranolol (a competitive antagonist that relaxes the receptor activation), in the presence of Gpp(NH)p (which provides a virtual absence of GTPase) and millimolar concentrations of Mg2+ (the rate of this inactivation is relatively fast); (4) increasing the concentration of Gpp(NH)p can saturate the steady-state activity of adenylate cyclase. The saturated enzyme activity was lower than that induced by isoprenaline under the same conditions. This additional agonist-induced activation was reversible. In the light of these results, we conclude that agonist can also activate the guanine nucleotide-saturated system in the absence of GTPase by a mechanism other than guanine nucleotide exchange. We explain these phenomena in the framework of a quinternary complex model as an agonist-induced and receptor-mediated dissociation of guanine nucleotide-saturated residual heterotrimer, the equilibrium concentration of which is not necessarily zero. These results, which suggest a continuous interaction between receptor and G-protein, can hardly be accommodated by the collision coupling model that was originally suggested for the present experimental system and then applied to many other G-protein systems. Therefore we

  14. Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels.

    PubMed

    Makarchikov, Alexander F; Wins, Pierre; Janssen, Edwin; Wieringa, Bé; Grisar, Thierry; Bettendorff, Lucien

    2002-10-21

    Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP+ATP if ThTP+ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase 1 (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase.

  15. Effects of Acetazolamide on the Unrinary Excretion of Cyclic AMP and on the Activity of Renal Adenyl Cyclase

    PubMed Central

    Rodriguez, Hector J.; Walls, John; Yates, Jesse; Klahr, Saulo

    1974-01-01

    Acetazolamide, an inhibitor of the enzyme carbonic anhydrase, increased the urinary excretion of cyclic AMP in normal and parathyroidectomized rats. The increase was greater in rats with intact parathyroid glands than in parathyroidectomized rats. This rise in the urinary excretion of cyclic AMP was not due to an increase in urine flow or a change in urine pH. Furosemide caused an increase in urine flow, but did not affect the excretion of cyclic AMP or phosphate. Alkalinization of the urine with bicarbonate did not increase the urinary excretion of phosphate or cyclic AMP. Acetazolamide increased the productionof cyclic AMP by rat renal cortical slices in vitro. This effect was dose-dependent. Acetazolamide also stimulated the activity of renal cortical adenyl cyclase in a dose-dependent manner but had no effect on the activity of cyclic nucleotide phosphodiesterase. The pattern of urinary excretion of cyclic AMP and phosphate after administration of acetazolamide was similar to that observed in rats given parathyroid hormone. It is suggested that acetazolamide stimulates the renal production of cyclic AMP by activating adenyl cyclase and that this may be the mechanism by which this inhibitor of carbonic anhydrase produces phosphaturia. PMID:4357608

  16. Crystal structure and substrate specificity of plant adenylate isopentenyltransferase from Humulus lupulus: distinctive binding affinity for purine and pyrimidine nucleotides.

    PubMed

    Chu, Hsing-Mao; Ko, Tzu-Ping; Wang, Andrew H-J

    2010-03-01

    Cytokinins are important plant hormones, and their biosynthesis most begins with the transfer of isopentenyl group from dimethylallyl diphosphate (DMAPP) to the N6-amino group of adenine by either adenylate isopentenyltransferase (AIPT) or tRNA-IPT. Plant AIPTs use ATP/ADP as an isopentenyl acceptor and bacterial AIPTs prefer AMP, whereas tRNA-IPTs act on specific sites of tRNA. Here, we present the crystal structure of an AIPT-ATP complex from Humulus lupulus (HlAIPT), which is similar to the previous structures of Agrobacterium AIPT and yeast tRNA-IPT. The enzyme is structurally homologous to the NTP-binding kinase family of proteins but forms a solvent-accessible channel that binds to the donor substrate DMAPP, which is directed toward the acceptor substrate ATP/ADP. When measured with isothermal titration calorimetry, some nucleotides displayed different binding affinities to HlAIPT with an order of ATP > dATP approximately ADP > GTP > CTP > UTP. Two basic residues Lys275 and Lys220 in HlAIPT interact with the beta and gamma-phosphate of ATP. By contrast, the interactions are absent in Agrobacterium AIPT because they are replaced by the acidic residues Asp221 and Asp171. Despite its structural similarity to the yeast tRNA-IPT, HlAIPT has evolved with a different binding strategy for adenylate.

  17. Properties of the separated catalytic and regulatory units of brain adenylate cyclase.

    PubMed Central

    Strittmatter, S; Neer, E J

    1980-01-01

    Adenylate cyclase from bovine brain cortex was solubilized with 14 mM cholate and 1 M (NH4)2SO4. Gel filtration over a column of Sepharose 6B separated the catalytic unit (CU) from a factor (G/F) that confers responsiveness to 5'-guanylyl imidophosphate (p[NH]ppG) or fluoride. The separated CU, which elutes with a Kav, of 0.48 +/- 0.01 (n=5), is not responsive to p[NH]ppG or fluoride and is relatively inactive when Mg . ATP is the substrate but activated 8-15-fold by Mn2+. The separated G/F elutes with a Kav of 0.70 +/- 0.02 (n=4). It restores the responsiveness of the CU to p[NH]ppG and fluoride. Activation of the enzyme by p[NH]ppG before solubilization does not decrease the amount of G/F eluting with a Kav of 0.7. Therefore, the G/F is probably present in brain cortex in excess over the CU. p[NH]ppG stabilizes the G/F but not the CU against thermal inactivation, suggesting that it interacts with G/F and not with CU. Incubation of the G/F with p[NH]ppG before addition of CU markedly increases the rate of activation of the reconstituted enzyme by p[NH]ppG. We propose, therefore, that the rate-limiting step in adenylate cyclase activation is a process in G/F alone and not a slow conformational change in CU or a slow association of G/F with CU. Binding of p[NH]ppG to the isolated G/F appears to be readily reversible; the ability of fully activated G/F to stimulate CU can be blocked if GDP is added before CU. In contrast, after the CU has been activated by interaction with G/F, GDP cannot reverse the activation. This suggests that association with the CU increases the affinity of G/F for p[NH]ppG. PMID:6935648

  18. Adenyl cyclase activator forskolin protects against Huntington's disease-like neurodegenerative disorders

    PubMed Central

    Mehan, Sidharth; Parveen, Shaba; Kalra, Sanjeev

    2017-01-01

    Long term suppression of succinate dehydrogenase by selective inhibitor 3-nitropropionic acid has been used in rodents to model Huntington's disease where mitochondrial dysfunction and oxidative damages are primary pathological hallmarks for neuronal damage. Improvements in learning and memory abilities, recovery of energy levels, and reduction of excitotoxicity damage can be achieved through activation of Adenyl cyclase enzyme by a specific phytochemical forskolin. In this study, intraperitoneal administration of 10 mg/kg 3-nitropropionic acid for 15 days in rats notably reduced body weight, worsened motor cocordination (grip strength, beam crossing task, locomotor activity), resulted in learning and memory deficits, greatly increased acetylcholinesterase, lactate dehydrogenase, nitrite, and malondialdehyde levels, obviously decreased adenosine triphosphate, succinate dehydrogenase, superoxide dismutase, catalase, and reduced glutathione levels in the striatum, cortex and hippocampus. Intragastric administration of forskolin at 10, 20, 30 mg/kg dose-dependently reversed these behavioral, biochemical and pathological changes caused by 3-nitropropionic acid. These results suggest that forskolin exhibits neuroprotective effects on 3-nitropropionic acid-induced Huntington's disease-like neurodegeneration. PMID:28400813

  19. Global Transitions of Proteins Explored by a Multiscale Hybrid Methodology: Application to Adenylate Kinase

    PubMed Central

    Gur, Mert; Madura, Jeffry D.; Bahar, Ivet

    2013-01-01

    Efficient and accurate mapping of transition pathways is a challenging problem in allosteric proteins. We propose here a to our knowledge new methodology called collective molecular dynamics (coMD). coMD takes advantage of the collective modes of motions encoded by the fold, simultaneously evaluating the interactions and energetics via a full-atomic MD simulation protocol. The basic approach is to deform the structure collectively along the modes predicted by the anisotropic network model, upon selecting them via a Monte Carlo/Metropolis algorithm from among the complete pool of all accessible modes. Application to adenylate kinase, an allosteric enzyme composed of three domains, CORE, LID, and NMP, shows that both open-to-closed and closed-to-open transitions are readily sampled by coMD, with large-scale motions of the LID dominating. An energy-barrier crossing occurs during the NMP movements. The energy barrier originates from a switch between the salt bridges K136-D118 at the LID-CORE interface and K57-E170 and D33-R156 at the CORE-NMP and LID-NMP interfaces, respectively. Despite its simplicity and computing efficiency, coMD yields ensembles of transition pathways in close accord with detailed full atomic simulations, lending support to its utility as a multiscale hybrid method for efficiently exploring the allosteric transitions of multidomain or multimeric proteins. PMID:24094405

  20. Riboswitch control of induction of aminoglycoside resistance acetyl and adenyl-transferases.

    PubMed

    He, Weizhi; Zhang, Xuhui; Zhang, Jun; Jia, Xu; Zhang, Jing; Sun, Wenxia; Jiang, Hengyi; Chen, Dongrong; Murchie, Alastair I H

    2013-08-01

    The acquisition of antibiotic resistance by human pathogens poses a significant threat to public health. The mechanisms that control the proliferation and expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are a historically important class of antibiotics that were introduced in the 1940s. Aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug or enzymatic modification of the target rRNA through methylation or through the overexpression of efflux pumps. In our recent paper, we reported that expression of the aminoglycoside resistance genes encoding the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes was controlled by an aminoglycoside-sensing riboswitch RNA. This riboswitch is embedded in the leader RNA of the aac/aad genes and is associated with the integron cassette system. The leader RNA can sense and bind specific aminoglycosides such that the binding causes a structural transition in the leader RNA, which leads to the induction of aminoglycoside antibiotic resistance. Specific aminoglycosides induce reporter gene expression mediated by the leader RNA. Aminoglycoside RNA binding was measured directly and, aminoglycoside-induced changes in RNA structure monitored by chemical probing. UV cross-linking and mutational analysis identified potential aminoglycoside binding sites on the RNA.

  1. Prokaryotic adenylate cyclase toxin stimulates anterior pituitary cells in culture

    SciTech Connect

    Cronin, M.J.; Evans, W.S.; Rogol, A.D.; Weiss, A.A.; Thorner, M.O.; Orth, D.N.; Nicholson, W.E.; Yasumoto, T.; Hewlett, E.L.

    1986-08-01

    Bordetella pertussis synthesis a variety of virulence factors including a calmodulin-dependent adenylate cyclase (AC) toxin. Treatment of anterior pituitary cells with this AC toxin resulted in an increase in cellular cAMP levels that was associated with accelerated exocytosis of growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH). The kinetics of release of these hormones, however, were markedly different; GH and prolactin were rapidly released, while LH and ACTH secretion was more gradually elevated. Neither dopamine agonists nor somatostatin changes the ability of AC toxin to generate cAMP (up to 2 h). Low concentrations of AC toxin amplified the secretory response to hypophysiotrophic hormones. The authors conclude that bacterial AC toxin can rapidly elevate cAMP levels in anterior pituitary cells and that it is the response that explains the subsequent acceleration of hormone release.

  2. Conformational transitions of Adenylate Kinase: switching by cracking

    PubMed Central

    Whitford, Paul C.; Miyashita, Osamu; Levy, Yaakov; Onuchic, José N.

    2007-01-01

    Conformational heterogeneity in proteins is known to often be the key to their function. We present a coarse grained model to explore the interplay between protein structure, folding and function which is applicable to allosteric or non-allosteric proteins. We employ the model to study the detailed mechanism of the reversible conformational transition of Adenylate Kinase (AKE) between the open to the closed conformation, a reaction that is crucial to the protein’s catalytic function. We directly observe high strain energy which appears to be correlated with localized unfolding during the functional transition. This work also demonstrates that competing native interactions from the open and closed form can account for the large conformational transitions in AKE. We further characterize the conformational transitions with a new measure ΦFunc, and demonstrate that local unfolding may be due, in part, to competing intra-protein interactions. PMID:17217965

  3. Structural characterization of Burkholderia pseudomallei adenylate kinase (Adk): Profound asymmetry in the crystal structure of the 'open' state

    SciTech Connect

    Buchko, G.W.; Robinson, H.; Abendroth, J.; Staker, B. L.; Myler, P. J.

    2010-04-16

    In all organisms adenylate kinases (Adks) play a vital role in cellular energy metabolism and nucleic acid synthesis. Due to differences in catalytic properties between the Adks found in prokaryotes and in the cytoplasm of eukaryotes, there is interest in targeting this enzyme for new drug therapies against infectious bacterial agents. Here we report the 2.1 {angstrom} resolution crystal structure for the 220-residue Adk from Burkholderia pseudomallei (BpAdk), the etiological agent responsible for the infectious disease melioidosis. The general structure of apo BpAdk is similar to other Adk structures, composed of a CORE subdomain with peripheral ATP-binding (ATP{sub bd}) and LID subdomains. The two molecules in the asymmetric unit have significantly different conformations, with a backbone RMSD of 1.46 {angstrom}. These two BpAdk conformations may represent 'open' Adk sub-states along the preferential pathway to the 'closed' substrate-bound state.

  4. Structural characterization of Burkholderia pseudomallei adenylate kinase (Adk): Profound asymmetry in the crystal structure of the ‘open’ state

    PubMed Central

    Buchko, Garry W.; Robinson, Howard; Abendroth, Jan; Staker, Bart L.; Myler, Peter J.

    2010-01-01

    In all organisms adenylate kinases (Adks) play a vital role in cellular energy metabolism and nucleic acid synthesis. Due to differences in catalytic properties between the Adks found in prokaryotes and in the cytoplasm of eukaryotes, there is interest in targeting this enzyme for new drugs therapies against infectious bacterial agents. Here we report the 2.1 Å resolution crystal structure for the 220-residue Adk from Burkholderia pseudomallei (BpAdk), the etiological agent responsible for the infectious disease meliodosis. The general structure of apo BpAdk is similar to other Adk structures, composed of a CORE subdomain with peripheral ATP-binding (ATPbd) and LID subdomains. The two molecules in the asymmetric unit have significantly different conformations, with a backbone RMSD of 1.46 Å. These two BpAdk conformations may represent ‘open’ Adk sub-states along the preferential pathway to the ‘closed’ substrate-bound state. PMID:20331978

  5. Covalent aspartylation of aspartyl-tRNA synthetase from Bakers' yeast by its cognat aspartyl adenylate: identification of the labeled residues

    SciTech Connect

    Mejdoub, H.; Kern, D.; Giege, R.; Ebel, J.P.; Boulanger, Y.; Reinbolt, J.

    1987-04-07

    Aspartyl-tRNA synthetase from bakers' yeast gives an unstable complex with the cognate adenylate, which reacts after dissociation with amino acid side chains of the protein. This leads to a covalent incorporation of (/sup 14/C)-aspartic acid into aspartyl-tRNA synthetase via amide or ester bonds formed between the ..cap alpha..-carboxyl group of activated aspartic acid and accessible lysines, serines, and threonines. This property is used to label the peptides at the surface of the enzyme. The main labeled residues have been identified, and their location in the primary structure is discussed in relation to structural properties of aspartyl-tRNA synthetase.

  6. Crystal structure of a trimeric archaeal adenylate kinase from the mesophile Methanococcus maripaludis with an unusually broad functional range and thermal stability.

    PubMed

    Davlieva, Milya; Shamoo, Yousif

    2010-02-01

    The structure of the trimeric adenylate kinase from the Archaebacteria Methanococcus mariplaludis (AK(MAR)) has been solved to 2.5-A resolution and the temperature dependent stability and kinetics of the enzyme measured. The K(M) and V(max) of AK(MAR) exhibit only modest temperature dependence from 30 degrees -60 degrees C. Although M. mariplaludis is a mesophile with a maximum growth temperature of 43 degrees C, AK(MAR) has a very broad functional range and stability (T(m) = 74.0 degrees C) that are more consistent with a thermophilic enzyme with high thermostability and exceptional activity over a wide range of temperatures, suggesting that this microbe may have only recently invaded a mesophilic niche and has yet to fully adapt. A comparison of the Local Structural Entropy (LSE) for AK(MAR) to the related adenylate kinases from the mesophile Methanococcus voltae and thermophile Methanococcus thermolithotrophicus show that changes in LSE are able to fully account for the intermediate stability of AK(MAR) and highlights a general mechanism for protein adaptation in this class of enzymes.

  7. Conformational heterogeneity within the LID domain mediates substrate binding to Escherichia coli adenylate kinase: function follows fluctuations.

    PubMed

    Schrank, Travis P; Wrabl, James O; Hilser, Vincent J

    2013-01-01

    Proteins exist as dynamic ensembles of molecules, implying that protein amino acid sequences evolved to code for both the ground state structure as well as the entire energy landscape of excited states. Accumulating theoretical and experimental evidence suggests that enzymes use such conformational fluctuations to facilitate allosteric processes important for substrate binding and possibly catalysis. This phenomenon can be clearly demonstrated in Escherichia coli adenylate kinase, where experimentally observed local unfolding of the LID subdomain, as opposed to a more commonly postulated rigid-body opening motion, is related to substrate binding. Because "entropy promoting" glycine mutations designed to increase specifically the local unfolding of the LID domain also affect substrate binding, changes in the excited energy landscape effectively tune the function of this enzyme without changing the ground state structure or the catalytic site. Thus, additional thermodynamic information, above and beyond the single folded structure of an enzyme-substrate complex, is likely required for a full and quantitative understanding of how enzymes work.

  8. Zipping and Unzipping of Adenylate Kinase: Atomistic Insights into the Ensemble of Open ↔ Closed Transitions

    PubMed Central

    Beckstein, Oliver; Denning, Elizabeth J.; Perilla, Juan R.; Woolf, Thomas B.

    2009-01-01

    Adenylate kinase (AdK), a phosphotransferase enzyme, plays an important role in cellular energy homeostasis. It undergoes a large conformational change between an open and a closed state, even in the absence of substrate. We investigate the apo-AdK transition at the atomic level both with free energy calculations and our new dynamic importance sampling (DIMS) molecular dynamics (MD) method. DIMS is shown to sample biologically relevant conformations as verified by comparing an ensemble of hundreds of DIMS transitions to AdK crystal structure intermediates. The simulations reveal in atomic detail how hinge regions partially and intermittently unfold during the transition. Conserved salt bridges are seen to have important structural and dynamic roles; in particular four ionic bonds are identified that open in a sequential, zipper-like fashion and thus dominate the free energy landscape of the transition. Transitions between the closed and open conformations only have to overcome moderate free energy barriers. Unexpectedly, the closed and open state encompass broad free energy basins that contain conformations differing in domain hinge motions by up to 40°. The significance of these extended states is discussed in relation to recent experimental FRET measurements. Taken together, these results demonstrate how a small number of cooperative key interactions can shape the overall dynamics of an enzyme and suggest an “all-or-nothing” mechanism for the opening and closing of AdK. Our efficient DIMS-MD computer simulation approach can provide a detailed picture of a functionally important macromolecular transition and thus help to interpret and suggest experiments to probe the conformational landscape of dynamic proteins such as AdK. PMID:19751742

  9. Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States.

    PubMed

    Rogne, Per; Wolf-Watz, Magnus

    2016-10-04

    Proteins are often functionally dependent on conformational changes that allow them to sample structural states that are sparsely populated in the absence of a substrate or binding partner. The distribution of such structural microstates is governed by their relative stability, and the kinetics of their interconversion is governed by the magnitude of associated activation barriers. Here, we have explored the interplay among structure, stability, and function of a selected enzyme, adenylate kinase (Adk), by monitoring changes in its enzymatic activity in response to additions of urea. For this purpose we used a (31)P NMR assay that was found useful for heterogeneous sample compositions such as presence of urea. It was found that Adk is activated at low urea concentrations whereas higher urea concentrations unfolds and thereby deactivates the enzyme. From a quantitative analysis of chemical shifts, it was found that urea redistributes preexisting structural microstates, stabilizing a substrate-bound open state at the expense of a substrate-bound closed state. Adk is rate-limited by slow opening of substrate binding domains and the urea-dependent redistribution of structural states is consistent with a model where the increased activity results from an increased rate-constant for domain opening. In addition, we also detected a strong correlation between the catalytic free energy and free energy of substrate (ATP) binding, which is also consistent with the catalytic model for Adk. From a general perspective, it appears that urea can be used to modulate conformational equilibria of folded proteins toward more expanded states for cases where a sizeable difference in solvent-accessible surface area exists between the states involved. This effect complements the action of osmolytes, such as trimethylamine N-oxide, that favor more compact protein states. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

    SciTech Connect

    Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.; Kunkel, Thomas A.; Williams, R. Scott

    2013-12-22

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing a ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA–DNA, and acting in an RNA–DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure–function studies of human APTX–RNA–DNA–AMP–Zn complexes define a mechanism for detecting and reversing adenylation at RNA–DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA–DNA may contribute to neurological disease.

  11. The influence of EDTA on adenylate cyclase activity in membranes from rat and mouse myocardium.

    PubMed

    Morris, S A; Bilezikian, J P

    1987-11-12

    Inclusion of EDTA in the homogenization buffer of both mouse and rat myocardium profoundly alters the properties of the adenylate cyclase complex. EDTA leads to an increase in the Vmax for adenylate cyclase activity due to all of the following agents: isoproterenol, Gpp[NH]p, forskolin and Mg2+. For forskolin and Mg2+, the EDTA-associated increase in Vmax is not accompanied by a change in sensitivity to activation. However, EDTA is associated with enhanced sensitivity to activation by isoproterenol and increased sensitivity to the effect of Mg2+ on isoproterenol-dependent adenylate cyclase activity. A result of greater isoproterenol-dependent adenylate cyclase activity, due to the presence of EDTA, is an attenuated synergistic contribution of Gpp[NH]p. Changes in stimulatable adenylate cyclase activity as a result of EDTA occurs in concert with effects of cholera toxin upon ADPribosylation of the guanine nucleotide regulatory protein, Ns. Substantial auto-ADP-ribosylation occurs in a cholera toxin-sensitive 42 kDa band in membranes prepared in the presence of EDTA. In addition, cofactor and substrate requirements in the cholera toxin-dependent ADP-ribosylation reaction depend on the method of membrane preparation. The results suggest that the integrity of the adenylate cyclase complex depends in part on the attention given to proteolysis that may be activated during the course of homogenization.

  12. Characterization of the norepinephrine-activation of adenylate cyclase suggests a role in memory affirmation pathways. Overexposure to epinephrine inactivates adenylate cyclase, a causal pathway for stress-pathologies.

    PubMed

    Bennun, Alfred

    2010-05-01

    Incubation with noradrenaline (norepinephrine) of isolated membranes of rat's brain corpus striatum and cortex, showed that ionic-magnesium (Mg(2+)) is required for the neurotransmitter activatory response of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1)], AC. An Mg(2+)-dependent response to the activatory effects of adrenaline, and subsequent inhibition by calcium, suggest capability for a turnover, associated with cyclic changes in membrane potential and participation in a short-term memory pathway. In the cell, the neurotransmitter by activating AC generates intracellular cyclic AMP. Calcium entrance in the cell inhibits the enzyme. The increment of cyclic AMP activates kinase A and their protein phosphorylating activity, allowing a long-term memory pathway. Hence, consolidating neuronal circuits, related to emotional learning and memory affirmation. The activatory effect relates to an enzyme-noradrenaline complex which may participate in the physiology of the fight or flight response, by prolonged exposure. However, the persistence of an unstable enzyme complex turns the enzyme inactive. Effect concordant, with the observation that prolonged exposure to adrenaline, participates in the etiology of stress triggered pathologies. At the cell physiological level AC responsiveness to hormones could be modulated by the concentration of chelating metabolites. These ones produce the release of free ATP(4-), a negative modulator of AC and the Mg(2+) activated insulin receptor tyrosine kinase (IRTK), thus, allowing an integration of the hormonal response of both enzymes by ionic controls. This effect could supersede the metabolic feedback control by energy charge. Accordingly, maximum hormonal response of both enzymes, to high Mg(2+) and low free ATP(4-), allows a correlation with the known effects of low caloric intake increasing average life expectancy.

  13. Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells

    PubMed Central

    Martín, César; Etxaniz, Asier; Uribe, Kepa B.; Etxebarria, Aitor; González-Bullón, David; Arlucea, Jon; Goñi, Félix M.; Aréchaga, Juan; Ostolaza, Helena

    2015-01-01

    Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of “toxin-coated bacteria” proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or “free” in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system. PMID:26346097

  14. Hydrolytic properties of phenylalanyl- and N-acetylphenylalanyl adenylate anhydrides

    NASA Technical Reports Server (NTRS)

    Lacey, J. C., Jr.; Mullins, D. W., Jr.; Senaratne, N.

    1984-01-01

    The hydrolysis of phenylalynyl- and N-acetylephenylalanyl adenylate anhydrides (AcPhe-AMP) is studied experimentally using a new spectrophotometric method. The hydrolysis process was analyzed at low concentrations (0.0001 M), constant temperature of 25 C, constant buffer concentration (0.05 M), and as a function of pH. It is found that while Phe-AMP is susceptible to attack by OH(-), AcPhe-AMP is susceptible to acid decomposition as well. At a pH of 4 to 8, Phe-AMP hydolyzes faster than AcPhe-AMP, but at pH less than four or greater than eight, the blocked form hydrolyzes faster. Both forms are attacked by H2O at the same rate. The rate laws for the various hydrolytic mechanisms and the activation energies for the hydrolyses at pH 7.1 are given in a table, and the possible relevance of the findings to the origin and evolution of the process of protein synthesis is discussed.

  15. Hydrolytic properties of phenylalanyl- and N-acetylphenylalanyl adenylate anhydrides

    NASA Technical Reports Server (NTRS)

    Lacey, J. C., Jr.; Mullins, D. W., Jr.; Senaratne, N.

    1984-01-01

    The hydrolysis of phenylalynyl- and N-acetylephenylalanyl adenylate anhydrides (AcPhe-AMP) is studied experimentally using a new spectrophotometric method. The hydrolysis process was analyzed at low concentrations (0.0001 M), constant temperature of 25 C, constant buffer concentration (0.05 M), and as a function of pH. It is found that while Phe-AMP is susceptible to attack by OH(-), AcPhe-AMP is susceptible to acid decomposition as well. At a pH of 4 to 8, Phe-AMP hydolyzes faster than AcPhe-AMP, but at pH less than four or greater than eight, the blocked form hydrolyzes faster. Both forms are attacked by H2O at the same rate. The rate laws for the various hydrolytic mechanisms and the activation energies for the hydrolyses at pH 7.1 are given in a table, and the possible relevance of the findings to the origin and evolution of the process of protein synthesis is discussed.

  16. Chirally selective, intramolecular interaction observed in an aminoacyl adenylate anhydride

    NASA Technical Reports Server (NTRS)

    Lacey, J. C., Jr.; Hall, L. M.; Mullins, D. W., Jr.; Watkins, C. L.

    1985-01-01

    The interaction between amino acids and nucleotide bases is studied. The proton NMR spectrum of N-acetylphenylalanyl-AMP-anhydride is analyzed H8 and H2 signals, two upfield signals of equal size, and five phenylalanine ring proton signals are observed in the spectrum; the upfield movement of the proton and the racemization of the N-acetyl L-phenylalanine material are examined. The differences in the position of the signals due to the diastereoisomers are investigated. The separation of the D and L amino acyl adenylates using HPLC is described. H-1 NMR spectra of the isomers are examined in order to determine which isomer displays the strongest interaction between the phenyl ring and the adenine ring. The spectra reveal that the L isomer shows the highest upfield change of both H8 and H2 signals. It is noted that the phenyl ring lies over C2 of the adenine ring with the phenyl meta and para protons extended past the adenine ring and the phenyl ortho protons.

  17. Analysis of the Linker Region Joining the Adenylation and Carrier Protein Domains of the Modular Non-Ribosomal Peptide Synthetases

    PubMed Central

    Miller, Bradley R.; Sundlov, Jesse A.; Drake, Eric J.; Makin, Thomas A.; Gulick, Andrew M.

    2014-01-01

    Non-Ribosomal Peptide Synthetases (NRPSs) are multi-modular proteins capable of producing important peptide natural products. Using an assembly-line process the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation-PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C-terminal sub-domain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. PMID:24975514

  18. NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors.

    PubMed

    Srivastava, Sandeep Kumar; Tripathi, Rama Pati; Ramachandran, Ravishankar

    2005-08-26

    DNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents.

  19. Regulation of cytochrome c oxidase by adenylic nucleotides. Is oxidative phosphorylation feedback regulated by its end-products?

    PubMed

    Beauvoit, B; Rigoulet, M

    2001-01-01

    Cytochrome c oxidase, which catalyzes an irreversible step of the respiratory chain, is one of the rate-controlling steps of oxidative phosphorylation on isolated mitochondria. The rate of electron transfer through the complex is primarily controlled by the associated thermodynamic forces, i.e., the span in redox potential between oxygen and cytochrome c and the protonmotive force. However, the electron flux also depends on the various kinetic effectors, including adenylic nucleotides. Although the number of binding sites for ATP and ADP on cytochrome oxidase is still a matter of debate, experiments performed on the solubilized and reconstituted enzyme provide strong functional evidence that the mammalian cytochrome c oxidase binds adenylic nucleotides on both sides of the inner membrane. These effects include modification in cytochrome c affinity, allosteric inhibition and changes in proton pumping efficiency. Immunological studies have pointed out the role of subunit IV and that of an ATP-binding protein, subunit VIa, in these kinetic regulations. In yeast, the role of the nuclear-encoded subunits in assembly and regulation of the cytochrome c oxidase has been further substantiated by using gene-disruption analysis. Using a subunit VIa-null mutant, the consequences of the ATP regulation on oxidative phosphorylation have been further investigated on isolated mitochondria. Taken together, the data demonstrate that there are multiple regulating sites for ATP on the yeast cytochrome oxidase with respect to the location (matrix versus cytosolic side), kinetic effect (activation versus inhibition) and consequence on the flow-force relationships. The question is therefore raised as to the physiological meaning of such feedback regulation of the respiratory chain by ATP in the control and regulation of cellular energy metabolism.

  20. Evidence for adenylate cyclase as a scaffold protein for Ras2-Ira interaction in Saccharomyces cerevisie.

    PubMed

    Colombo, Sonia; Paiardi, Chiara; Pardons, Katrien; Winderickx, Joris; Martegani, Enzo

    2014-05-01

    Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.

  1. 2-Aryl-8-aza-3-deazaadenosine Analogues of 5’-O-[N-(Salicyl)sulfamoyl]adenosine: Nucleoside Antibiotics that Block Siderophore Biosynthesis in Mycobacterium tuberculosis

    PubMed Central

    Krajczyk, Anna; Zeidler, Joanna; Januszczyk, Piotr; Dawadi, Surendra; Boshoff, Helena I.; Barry, Clifton E.; Ostrowski, Tomasz; Aldrich, Courtney C.

    2016-01-01

    A series of 5’-O-[N-(salicyl)sulfamoyl]-2-aryl-8-aza-3-deazaadenosines were designed to block mycobactin biosynthesis in Mycobacterium tuberculosis (Mtb) through inhibition of the essential adenylating enzyme MbtA. The synthesis of the 2-aryl-8-aza-3-deazaadenosine nucleosides featured sequential copper-free palladium-catalyzed Sonogashira coupling of a precursor 4-cyano-5-iodo-1,2,3-triazolonucleoside with terminal alkynes and Minakawa-Matsuda annulation reaction. These modified nucleosides were shown to inhibit MbtA with apparent Ki values ranging from 6.1 to 25 nM and to inhibit Mtb growth under iron-deficient conditions with minimum inhibitory concentrations ranging from 12.5 to >50 μM. PMID:27265685

  2. Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

    PubMed Central

    Ohki, Mio; Sugiyama, Kanako; Kawai, Fumihiro; Tanaka, Hitomi; Nihei, Yuuki; Unzai, Satoru; Takebe, Masumi; Matsunaga, Shigeru; Adachi, Shin-ichi; Shibayama, Naoya; Zhou, Zhiwen; Koyama, Ryuta; Takahashi, Tetsuo; Tame, Jeremy R. H.; Iseki, Mineo; Park, Sam-Yong

    2016-01-01

    Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit. PMID:27247413

  3. Adenylate cyclase of human articular chondrocytes. Responsiveness to prostaglandins and other hormones.

    PubMed Central

    Houston, J P; McGuire, M K; Meats, J E; Ebsworth, N M; Russell, R G; Crawford, A; Mac Neil, S

    1982-01-01

    Adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] was shown to be present in cultured human articular chondrocytes. Optimal conditions of incubation time, protein and substrate concentrations and pH were determined in whole cell lysates. Maximal activity occurred at pH 8.5 with no decrease in activity up to pH 10.0. Adenylate cyclase activity of particulate membrane preparations was enhanced by the addition of crude cytosol preparations. The prostaglandins E1, E2, F1 alpha, F2 alpha, D2, B1, B2, A1 and A2, as well as adrenaline and isoprenaline, stimulated adenylate cyclase derived from either adult or foetal chondrocytes. No significant stimulation was observed in the presence of human calcitonin or glucagon. Bovine parathyroid hormone always significantly stimulated the adenylate cyclase derived from foetal chondrocytes, but not from adult chondrocytes. Preincubation of the chondrocytes in culture with indomethacin and with or without supernatant medium from cultured mononuclear cells increased the responsiveness of the adenylate cyclase to prostaglandin E1. PMID:7159397

  4. [Participation of adenylate kinase in the regulation of chloroplasts energy exchange].

    PubMed

    Kartashev, I M

    2009-01-01

    The influence of adenylate kinase on the rates of glucose-6-phosphate synthesis and ferricyanide reduction in a system containing chloroplasts, hexokinase, and ADP at low concentration during photophosphorylation has been studied. It has been found that the addition of adenylate kinase into the reaction medium under phosphorylation results in a simultaneous increase in the rate of ferricyanide reduction and glucose-6-phosphate synthesis. In this case, the ratio of glucose-6-phosphate formed to the quantity of ferricyanide reduced was close to unity as the concentration of adenylate kinase in the medium increased. The concentrations of glucose-6-phosphate and ferricyanide reduced in the system sharply increased with time; at the same time, no significant decrease in ADP concentration and AMP accumulation by the methods available was found. Hence, the limiting factors in these reactions are not the concentrations but the rates of diffusion of the substrates. Presumably, diffusion limitations in the system are eliminated owing to the participation of adenylate kinase. The results obtained are discussed in terms of the model according to which the regulation of the diffusion of adenine nucleotides and the control of regeneration of ATP according to its requirements in correlation with other regulation mechanisms can occur in chloroplasts upon adenylate kinase functioning by direct and reverse connection of the shuttle type.

  5. Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing

    PubMed Central

    Dzeja, Petras; Terzic, Andre

    2009-01-01

    Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network. PMID:19468337

  6. Interaction of the antiarrhythmic agents SR 33589 and amiodarone with the beta-adrenoceptor and adenylate cyclase in rat heart.

    PubMed Central

    Chatelain, P.; Meysmans, L.; Mattéazzi, J. R.; Beaufort, P.; Clinet, M.

    1995-01-01

    1. The effects of SR 33589 and amiodarone on the cardiac beta-adrenoceptor were studied in vitro and after chronic treatment by means of [125I]-(-)-iodocyanopindolol ([125I]-(-)-CYP) binding and measurement of adenylate cyclase activity. 2. Binding of [125I]-(-)-CYP was inhibited in a dose-dependent manner by SR 33589 (IC50=1.8 +/- 0.4 microM, nH=0.93 +/- 0.06) and amiodarone (IC50=8.7 +/- 2.0 microM, nH=9.2 +/- 0.03). Saturation binding experiments indicated a non-competitive interaction such that SR 33589 (1 and 3 microM) and amiodarone (5 and 10 microM) reduced the Bmax of [125I]-(-)-CYP binding without any effect on the KD. Kinetic studies showed that the rate of association of [125I]-(-)-CYP was unchanged while the rate of dissociation was increased both in the presence of SR 33589 (10 microM) and amiodarone (30 microM).3. Under the same conditions, the receptor stimulated adenylate cyclase activity was inhibited in a dose-dependent, but non-competitive manner, by SR 33589 (isoprenaline-, glucagon- and secretin-stimulated enzyme inhibited 50% at 6.8 +/- 0.6 microM, 31 +/- 10 microM and 12 +/- 3 microM, respectively) while the basal, GTP- and GPP(NH)p-stimulated enzyme was inhibited by 5-10% and the NaF and forskolin-stimulated enzyme by 50% at 500 microM. Amiodarone exhibited a similar pattern of inhibition. 4. After chronic oral treatment (50, 100, 150 mg kg(-1) per day, 14 days), both SR 33589 and amiodarone produced a dose-dependent decrease in Bmax without any effect on KD as determined from [125I]-(-)-CYP saturation experiments and a decrease of the isoprenaline- and glucagon-stimulated adenylate cyclase activity without any effect on basal enzyme activity or activity when stimulated by agents acting directly on regulatory catalytic units. 5. Unlike amiodarone, SR 33589 does not contain iodine substituents. Plasma levels of T3, T4, and rT3 were changed after SR 33589 treatment except a decrease in T4 level at the highest dose whilst the T4 T3 ratio and

  7. Crystal structure of the human ubiquitin-activating enzyme 5 (UBA5) bound to ATP: mechanistic insights into a minimalistic E1 enzyme.

    PubMed

    Bacik, John-Paul; Walker, John R; Ali, Mohsin; Schimmer, Aaron D; Dhe-Paganon, Sirano

    2010-06-25

    E1 ubiquitin-activating enzymes (UBAs) are large multidomain proteins that catalyze formation of a thioester bond between the terminal carboxylate of a ubiquitin or ubiquitin-like modifier (UBL) and a conserved cysteine in an E2 protein, producing reactive ubiquityl units for subsequent ligation to substrate lysines. Two important E1 reaction intermediates have been identified: a ubiquityl-adenylate phosphoester and a ubiquityl-enzyme thioester. However, the mechanism of thioester bond formation and its subsequent transfer to an E2 enzyme remains poorly understood. We have determined the crystal structure of the human UFM1 (ubiquitin-fold modifier 1) E1-activating enzyme UBA5, bound to ATP, revealing a structure that shares similarities with both large canonical E1 enzymes and smaller ancestral E1-like enzymes. In contrast to other E1 active site cysteines, which are in a variably sized domain that is separate and flexible relative to the adenylation domain, the catalytic cysteine of UBA5 (Cys(250)) is part of the adenylation domain in an alpha-helical motif. The novel position of the UBA5 catalytic cysteine and conformational changes associated with ATP binding provides insight into the possible mechanisms through which the ubiquityl-enzyme thioester is formed. These studies reveal structural features that further our understanding of the UBA5 enzyme reaction mechanism and provide insight into the evolution of ubiquitin activation.

  8. Crystal Structure of the Human Ubiquitin-activating Enzyme 5 (UBA5) Bound to ATP Mechanistic Insights into a Minimalistic E1 Enzyme

    SciTech Connect

    Bacik, John-Paul; Walker, John R.; Ali, Mohsin; Schimmer, Aaron D.; Dhe-Paganon, Sirano

    2010-08-30

    E1 ubiquitin-activating enzymes (UBAs) are large multidomain proteins that catalyze formation of a thioester bond between the terminal carboxylate of a ubiquitin or ubiquitin-like modifier (UBL) and a conserved cysteine in an E2 protein, producing reactive ubiquityl units for subsequent ligation to substrate lysines. Two important E1 reaction intermediates have been identified: a ubiquityl-adenylate phosphoester and a ubiquityl-enzyme thioester. However, the mechanism of thioester bond formation and its subsequent transfer to an E2 enzyme remains poorly understood. We have determined the crystal structure of the human UFM1 (ubiquitin-fold modifier 1) E1-activating enzyme UBA5, bound to ATP, revealing a structure that shares similarities with both large canonical E1 enzymes and smaller ancestral E1-like enzymes. In contrast to other E1 active site cysteines, which are in a variably sized domain that is separate and flexible relative to the adenylation domain, the catalytic cysteine of UBA5 (Cys{sup 250}) is part of the adenylation domain in an {alpha}-helical motif. The novel position of the UBA5 catalytic cysteine and conformational changes associated with ATP binding provides insight into the possible mechanisms through which the ubiquityl-enzyme thioester is formed. These studies reveal structural features that further our understanding of the UBA5 enzyme reaction mechanism and provide insight into the evolution of ubiquitin activation.

  9. Localization of nigrostriatal dopamine receptor subtypes and adenylate cyclase

    SciTech Connect

    Filloux, F.; Dawson, T.M.; Wamsley, J.K.

    1988-04-01

    Quantitative autoradiography using (/sup 3/H)-SCH 23390, (/sup 3/H)-sulpiride and (/sup 3/H)-forskolin was used to assess the effects of single and combined neurotoxin lesions of the nigrostriatal pathway in the rat brain on dopamine (DA) receptor subtypes and adenylate cyclase (AC), respectively. Ibotenic acid (IA) lesions of the caudate-putamen (CPu) resulted in near total loss of both (/sup 3/H)-SCH 23390 and of (/sup 3/H)-forskolin binding in the ipsilateral CPu and substantia nigra reticulata (SNR). (/sup 3/H)-sulpiride binding in the CPu was only partially removed by this same lesion, and nigral (/sup 3/H)-sulpiride binding was virtually unchanged. 6-Hydroxydopamine (6-OHDA) and IA lesions of the substantia nigra compacta (SNC) did not affect (/sup 3/H)-SCH 23390 or (/sup 3/H)-forskolin binding, but largely removed (/sup 3/H)-sulpiride binding in the SNC. A 6-OHDA lesion of the nigrostriatal pathway followed by an ipsilateral IA injection of the CPu failed to further reduce (/sup 3/H)-sulpiride binding in the CPu. These results demonstrate that postsynaptic DA receptors in the CPu are of both the D1 and D2 variety; however, a portion of D2 receptors in the CPu may be presynaptic on afferent nerve terminals to this structure. D1 receptors in the SNR are presynaptic on striatonigral terminals, whereas the D2 receptors of the SNC are autoreceptors on nigral DA neurons. The existence of presynaptic D2 receptors on nigrostriatal DA-ergic terminals could not be confirmed by this study. Co-localization of D1 receptors and AC occurs in both the CPu and SNR.

  10. Adenylate cyclase regulates elongation of mammalian primary cilia

    SciTech Connect

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; Hoorn, Frans A. van der

    2009-10-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3{beta} by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

  11. The crystal structure of Mycobacterium tuberculosis adenylate kinase in complex with two molecules of ADP and Mg2+ supports an associative mechanism for phosphoryl transfer

    PubMed Central

    Bellinzoni, Marco; Haouz, Ahmed; Graña, Martin; Munier-Lehmann, Hélène; Shepard, William; Alzari, Pedro M.

    2006-01-01

    The crystal structure of Mycobacterium tuberculosis adenylate kinase (MtAK) in complex with two ADP molecules and Mg2+ has been determined at 1.9 Å resolution. Comparison with the solution structure of the enzyme, obtained in the absence of substrates, shows significant conformational changes of the LID and NMP-binding domains upon substrate binding. The ternary complex represents the state of the enzyme at the start of the backward reaction (ATP synthesis). The structure is consistent with a direct nucleophilic attack of a terminal oxygen from the acceptor ADP molecule on the β-phosphate from the donor substrate, and both the geometry and the distribution of positive charge in the active site support the hypothesis of an associative mechanism for phosphoryl transfer. PMID:16672241

  12. The ice worm, Mesenchytraeus solifugus, elevates adenylate levels at low physiological temperature.

    PubMed

    Napolitano, Michael J; Nagele, Robert G; Shain, Daniel H

    2004-01-01

    The ice worm, Mesenchytraeus solifugus, is among a few metazoan species that survive exclusively in glacier ice/snow. In this study, we demonstrate that ice worm adenylate levels [i.e. adenosine 5'-triphosphate (ATP), ADP and AMP] are maintained at levels well above their mesophilic counterparts, and that their response to temperature change is distinctly opposite, namely, ice worms increase energy levels as temperatures fall. Initially, this response is characterized by a sharp spike in [ATP] and the adenylate energy charge (even at sub-zero temperatures), which is followed by corresponding increases in [ADP] and [AMP] within a few days. These results suggest that ice worms have evolved a compensatory mechanism by which gains in adenylate nucleotides off-set, at least in part, the inherent lethargy and death usually associated with cold temperature.

  13. Synthesis of amino acyl adenylates using the tert-butoxycarbonyl protecting group

    NASA Technical Reports Server (NTRS)

    Armstrong, D. W.; Seguin, R.; Saburi, M.; Fendler, J. H.

    1979-01-01

    The synthesis of amino acyl adenylates using N-tert-butoxycarbonyl-protected amino acids is reported. Anhydrous solutions containing N-tert-butoxycarbonyl alanine, phenylalanine, and methionine were combined with the anhydrous mono (tri-n-octylammonium) salt of adenosine 5'-phosphate and the resultant amino acyl adenylates were characterized by means of elemental analysis, and infrared and proton NMR spectroscopy. Amino acyl adenylate yields of up to 60% were obtained with high purity at room temperatures. The reported synthesis is considered to represent a large improvement over previous methods due to the purity of the products, normal temperature requirements, and the stability of the starting compounds, which suggests its use in investigations of prebiotic oligo- and polypeptide synthesis.

  14. Distribution of adenylate cyclase and GTP-binding proteins in hepatic plasma membranes.

    PubMed

    Dixon, B S; Sutherland, E; Alexander, A; Nibel, D; Simon, F R

    1993-10-01

    Hepatic membrane subfractions prepared from control rats demonstrated forskolin (FSK)-stimulated adenylate cyclase activity in the basolateral (sinusoidal) but not apical (canalicular) plasma membrane. After bile duct ligation (BDL) for 12 or 24 h, there was an increase in FSK-stimulated adenylate cyclase activity in the apical membrane (54.2 +/- 3.9 pmol.mg-1 x min-1). The mechanism for this increase was explored further. ATP hydrolysis was found to be much higher in the apical than the basolateral membrane. Increasing the ATP levels in the assay enhanced apical membrane adenylate cyclase activity (10.5 +/- 0.2 pmol.mg-l.min-1); however, total adenosinetriphosphatase (ATPase) activity was not altered after BDL. Extraction of the apical membrane with bile acids or other detergents resulted in a two- to threefold increase in adenylate cyclase activity (30.6 +/- 3.6 pmol.mg-1 x min-1; detergent C12E8) This suggested that bile duct ligation was acting via the detergent-like action of bile acids to uncover latent adenylate cyclase activity on apical membranes. Further studies demonstrated that both BDL and detergent extraction also enhanced toxin-directed ADP-ribosylation of Gs alpha (cholera toxin) and Gi alpha (pertussis toxin) in the apical but not the basolateral membrane. After BDL, Gi alpha was found to be twofold greater in the apical membrane than the basolateral membrane. Immunoblotting using specific G protein antibodies further confirmed that apical membranes from control rats had a higher concentration of Gi1, 2 alpha and beta and slightly elevated levels of Gi3 alpha and Gs alpha compared with the basolateral membrane. The results demonstrate that adenylate cyclase and heterotrimeric GTP-binding proteins are present on the apical membrane, but measurement of their functional activity requires detergent permeabilization of apical membrane vesicles and is limited by the presence of high ATPase activity.

  15. Clay catalyzed polymerization of amino acid adenylates and its relationship to biochemical reactions

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1978-01-01

    The adsorption and polymerization of alanine adenylate on montmorillonite at pH 7 when either its interspacial faces or its edger are blocked by an excess of histidine or sodium hexametaphosphate was investigated. Results indicate that alanine adenylate can be adsorbed any place on the interspacial spaces of the clay; however, adsorption of its phosphate part, which is limited to the edges of the clay, is necessary for polymerization to occur. As a result, polymerization takes place only at sites on the interspacial faces bordering the edges.

  16. [Characteristics of interaction of adenylate cyclase modulators and phosphoinositide cell signaling systems with lipid langmuir monolayers].

    PubMed

    Liakhov, O M; Prokopenko, V V; Prokopenko, R A; Mohylevych, S Ie

    2006-01-01

    Interaction of two groups of bioregulators, which oppositely affect activity of adenylate cyclase and phosphoinositide cellular signaling systems, with the Langmuir monolayer films made of natural lecithin was studied. Most significant influence on the structural and energy characteristics of lipid monolayers was revealed for the group of bioregulators, which inhibit polyphosphoinositide signaling system or/and activate adenylate cyclase signaling system. It is shown, that using the cluster analysis the bioregulators can be divided into two groups according to general orientation of their action on the considered systems of transduction of a signal.

  17. Clay catalyzed polymerization of amino acid adenylates and its relationship to biochemical reactions

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1978-01-01

    The adsorption and polymerization of alanine adenylate on montmorillonite at pH 7 when either its interspacial faces or its edger are blocked by an excess of histidine or sodium hexametaphosphate was investigated. Results indicate that alanine adenylate can be adsorbed any place on the interspacial spaces of the clay; however, adsorption of its phosphate part, which is limited to the edges of the clay, is necessary for polymerization to occur. As a result, polymerization takes place only at sites on the interspacial faces bordering the edges.

  18. Inhibition of forskolin-stimulated adenylate cyclase activity by 5-HT receptor agonists.

    PubMed

    Devivo, M; Maayani, S

    1985-12-17

    We measured the inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig hippocampal membranes by 5-HT, 5-carboxamidotryptamine (CAT) and 8-hydroxy-2-(di-n-propylamino) tetralin (PAT). Low concentrations of these agonists inhibited forskolin-stimulated adenylate cyclase activity in a concentration-dependent and saturable manner. The antagonist spiperone shifted the concentration-response curve to CAT to the right in a parallel manner. The EC50 values of CAT, PAT and 5-HT and the KB of spiperone suggest that this receptor may correspond to the 5-HT1A binding site.

  19. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-01-01

    The spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay, is studied. It is found that the degrees of polymerization of the oligopeptides and polypeptides obtained is dependent on the amounts of polypeptides that were preadsorbed. It is concluded that a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances.

  20. [The aspects of adenylate cyclase activity regulation in myocardium cell membranes during hypokinesia].

    PubMed

    Bulanova, K Ia; Komar, E S; Lobanok, L M

    1999-01-01

    Nonstimulated and isoproterenol, GTF, GITF, NaF stimulated activities of the adenylate cyclase in sarcolemma in white rats' myocardium was studied after two weeks of hypokinesia. As was established, in restrained animals the sensitivity of adenylate cyclase to the specified agents was increased and transition to the bimodal GTF regulation took place. It is hypothesised that involvement of membrane-bound Gi-proteins in the adrenergic effects on cardiomyocytes is one of mechanisms of the cardiotropic effects of restraint and heart distresses.

  1. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-01-01

    The spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay, is studied. It is found that the degrees of polymerization of the oligopeptides and polypeptides obtained is dependent on the amounts of polypeptides that were preadsorbed. It is concluded that a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances.

  2. Enzyme distribution in Pseudomonas aeruginosa.

    PubMed

    CAMPBELL, J J; HOGGLA; STRASDINE, G A

    1962-05-01

    Campbell, J. J. R. (The University of British Columbia, Vancouver, B.C., Canada), Loretta A. Hogg, and G. A. Strasdine. Enzyme distribution in Pseudomonas aeruginosa. J. Bacteriol. 83:1155-1160. 1962.-Previous studies on the distribution of enzymes in bacteria have indicated that, although individual enzymes were predominantly associated with a particular cellular structure, nevertheless some of the enzyme appeared to be present in all cellular fractions. In the present work with Pseudomonas aeruginosa, it was shown that, in general, an enzyme is present in only one cellular component. Hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, gluconic dehydrogenase, malic dehydrogenase, fumarase, isocitric dehydrogenase, isocitritase, and catalase were detected only in the soluble cytoplasm of the cell. Glucose oxidase and succinic dehydrogenase were detected only in the "ghost" fraction. Diphosphopyridine nucleotide oxidase was present in both "ghost" and ribosomal fractions but was most concentrated in the "ghost". Although adenylic kinase was found to be present in all fractions, it was possible to fractionate cells so that almost all of the activity was associated with the soluble cytoplasm a minor amount being associated with the "ghost." Adenosine triphosphatase was most concentrated in the "ghost" but appreciable activity appeared in the cytoplasm. Polynucleotide phosphorylase appeared to be the only enzyme that was convincingly associated with the ribosomes. However, a small amount of activity was associated with the soluble cytoplasm and with the "ghosts."

  3. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    PubMed

    Fry, D C; Kuby, S A; Mildvan, A S

    1986-02-01

    The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.

  4. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    PubMed Central

    Fry, D C; Kuby, S A; Mildvan, A S

    1986-01-01

    The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme. Images PMID:2869483

  5. Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.

    PubMed

    Ruan, Qiaoqiao; Chen, Yan; Gratton, Enrico; Glaser, Michael; Mantulin, William W

    2002-12-01

    Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the

  6. Dependence of the hormonal stimulation of adenylate cyclase on the fraction of the plasma membrane accessible for lateral displacement of proteins of the adenylate cyclase complex

    SciTech Connect

    Kazarov, A.R.; Rozenkrants, A.A.; Sobolev, A.S.

    1986-09-10

    Hormonal activation of the adenylate cyclase complex is associated with lateral displacement in the membrane of the proteins that constitute this complex. In this work an experimental investigation was made of the changes in the interaction of the proteins of the adenylate cyclase complex with the changing fraction of fluid lipids in the cell membrane. A decrease in the fraction of fluid lipids of rat reticulocyte membranes led to a decrease (all the way down to a total suppression) of the interaction of the ..beta..-adrenoreceptors with the regulatory N-proteins. The interaction of the N-proteins with the catalytic proteins was also suppressed. On the other hand, an increase in the fraction of fluid lipids led to more effective interaction. It was shown that in this case the functional intactness of the interacting proteins is unimpaired. An analysis of the results obtained, performed on the basis of the percolation theory, suggests the conclusion that the hormonal stimulation of adenylate cyclase depends on the fraction of fluid lipids in the membrane, and the proteins are displaced during interaction over distances comparable with the size of the membrane itself. It was also shown that characteristic activity of the ..beta..-agonist 1-isoproterenol varies from 1.0 to 0, depending on the fraction of fluid lipids in the membrane. The data obtained suggest that in the absence of guanylic nucleotides in the membrane in vitro there are no preexisting complexes with a high affinity for the agonist.

  7. In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model.

    PubMed

    Kim, H J; Nishikawa, S; Tokutomi, Y; Takenaka, H; Hamada, M; Kuby, S A; Uesugi, S

    1990-02-06

    Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.

  8. Modulation of Adenylate Energy Charge During the Swarmer Cycle of Hyphomicrobium neptunium

    PubMed Central

    Emala, Mary A.; Weiner, Ronald M.

    1983-01-01

    Adenylate energy charge was measured in the budding bacterium Hyphomicrobium neptunium through the course of the swarmer cycle. The energy charge was modulated, being low in swarm cells (0.64) and in cells initiating bud formation (0.57), an event which coincides with a round of DNA replication. PMID:6826528

  9. LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

    PubMed

    Clayton, R N; Shakespear, R A; Marshall, J C

    1978-06-01

    Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

  10. Subtyping of Salmonella enterica subspecies I using single nucleotide polymorphisms in adenylate cyclase (cyaA)

    USDA-ARS?s Scientific Manuscript database

    Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single nucleotide polymorphisms (SNPs) were characterized within adenylate cyclas...

  11. Hyaluronic acid as capacitation inductor: metabolic changes and membrane-associated adenylate cyclase regulation.

    PubMed

    Fernández, S; Córdoba, M

    2014-12-01

    The aim of this research was to study the effect of hyaluronic acid on bovine cryopreserved spermatozoa compared with heparin as regards the variation of capacitation induction, cellular oxidative metabolism and intracellular signal induced by membrane-associated adenylate cyclase to propose hyaluronic acid as a capacitation inductor. Heparin or hyaluronic acid and lysophosphatidylcholine were used to induce sperm capacitation and acrosome reaction, respectively. 2',5'-dideoxyadenosine was used as a membrane-associated adenylate cyclase inhibitor. The highest percentages of capacitated spermatozoa and live spermatozoa with acrosome integrity were obtained by incubating sperm for 60 min using 1000 μg/ml hyaluronic acid. In these conditions, capacitation induced by hyaluronic acid was lower compared with heparin; nonetheless both glycosaminoglycans promote intracellular changes that allow true acrosome reaction in vitro induced by lysophosphatidylcholine in bovine spermatozoa. Oxygen consumption in heparin-capacitated spermatozoa was significantly higher than in hyaluronic acid-treated spermatozoa. With all treatments, mitochondrial coupling was observed when a specific uncoupler of the respiratory chain was added. The inhibition of membrane-associated adenylate cyclase significantly blocked capacitation induction produced by hyaluronic acid, maintaining a basal sperm oxygen uptake in contrast to heparin effect in which both sperm parameters were inhibited, suggesting that the membrane-associated adenylate cyclase activation is involved in the intracellular signal mechanisms induced by both capacitation inductors, but only regulates mitochondrial oxidative phosphorylation in heparin-capacitated spermatozoa.

  12. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure

    SciTech Connect

    Scarpace, P.J.; Baresi, L.A.; Morley, J.E. Univ. of California, Los Angeles )

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the {beta}-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the {beta}-adrenergic pathway, adenylate cyclase activity and {beta}-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. {beta}-Adrenergic receptors were identified in BAT using ({sup 125}I)iodocyanopindolol. Binding sites had the characteristics of mixed {beta}{sub 1}- and {beta}{sub 2}-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in {beta}-adrenergic receptor density due to a loss of the {beta}{sub 1}-adrenergic subtype. This BAT {beta}-adrenergic receptor downregulation was tissue specific, since myocardial {beta}-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of {beta}-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability.

  13. Comparative effect of methioninyl adenylate on the growth of Salmonella typhimurium and Pseudomonas aeruginosa.

    PubMed

    Enouf, J; Laurence, F; Farrugia, G; Blanchard, P; Robert-Gero, M

    1976-10-11

    The bacteriostatic effect of methioninyl adenylate(MAMP)--a specific inhibitor of the enzyme methionyl-tRNA synthetase--was investigated on Salmonella typhimurium and Pseudomonas aeruginosa. 0.1 mM of this molecule added to the culture, inhibits the growth of S. typhimurium. The inhibition is specifically reversible by 0.1 mM L-methionine. In the same conditions even 1-2 mM MAMP has a very slight effect on the growth rate of P. aeruginosa and only during the first two generations. The same observation was made with the two other members of the fluorescens group P.fluorescens and P.putida. The growth rate of P. testosteroni with 1 mM MAMP in the medium is similar to the growth rate of P. aeruginosa but the other member of the acidovorans group P. acidovorans is much more affected by the smae concentration of the inhibitor. --P. multivorans is inhibited by MAMP like P. acidovorans but with a somewhat higher yield at the end of the culture. --MAMP has no effect on P. alcaligenes. The possible reasons for the weak bacteriostatic effect of MAMP on P. aeruginosa were investigated. It was established that the inhibitor enters the cells and is not used as a carbon and energy source. The intracellular methionine concentration in S. typhimurium and in P. aeruginosa is about the same and does not increase when bacteria are cultivated with MAMP. The MTS of the two microorganisms is inhibited by MAMP in vitro to about the same extent. Furthermore the tRNAmet from P. aeruginosa are fully acylated after 3 to 4 generations with this compound. Nevertheless MAMP elicits higher MTS activity in P. aeruginosa and in P. acidovorans after 1 h of incubation. The most striking difference between S. typhimurium and P. aeruginosa is that the intra and extracellular level of 5'phosphodiesterase which degrades MAMP is 10-20 fold higher in the second than in the first species.

  14. Metabolomic analysis of human oral cancer cells with adenylate kinase 2 or phosphorylate glycerol kinase 1 inhibition

    PubMed Central

    Ji, Eoon Hye; Cui, Li; Yuan, Xiaoqing; Cheng, Siliangyu; Messadi, Diana; Yan, Xinmin; Hu, Shen

    2017-01-01

    The purpose of this study was to use liquid chromatography-mass spectrometry (LC-MS) with XCMS for a quantitative metabolomic analysis of UM1 and UM2 oral cancer cells after knockdown of metabolic enzyme adenylate kinase 2 (AK2) or phosphorylate glycerol kinase 1 (PGK1). UM1 and UM2 cells were initially transfected with AK2 siRNA, PGK1 siRNA or scrambled control siRNA, and then analyzed with LC-MS for metabolic profiles. XCMS analysis of the untargeted metabolomics data revealed a total of 3200-4700 metabolite features from the transfected UM1 or UM2 cancer cells and 369-585 significantly changed metabolites due to AK2 or PGK1 suppression. In addition, cluster analysis showed that a common group of metabolites were altered by AK2 knockdown or by PGK1 knockdown between the UM1 and UM2 cells. However, the set of significantly changed metabolites due to AK2 knockdown was found to be distinct from those significantly changed by PGK1 knockdown. Our study has demonstrated that LC-MS with XCMS is an efficient tool for metabolomic analysis of oral cancer cells, and knockdown of different genes results in distinct changes in metabolic phenotypes in oral cancer cells. PMID:28243334

  15. Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis.

    PubMed

    Eby, Joshua C; Gray, Mary C; Warfel, Jason M; Merkel, Tod J; Hewlett, Erik L

    2017-01-01

    Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination. Copyright © 2017 American Society for Microbiology.

  16. The Pseudomonas aeruginosa Chp Chemosensory System Regulates Intracellular cAMP Levels by Modulating Adenylate Cyclase Activity

    PubMed Central

    Fulcher, Nanette B.; Holliday, Phillip M.; Klem, Erich; Cann, Martin J.; Wolfgang, Matthew C.

    2010-01-01

    Summary Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signaling molecule adenosine 3’, 5’-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems. PMID:20345659

  17. A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy.

    PubMed

    Hass, Mathias A S; Liu, Wei-Min; Agafonov, Roman V; Otten, Renee; Phung, Lien A; Schilder, Jesika T; Kern, Dorothee; Ubbink, Marcellus

    2015-02-01

    NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements.

  18. Relationship between bacterial virulence and nucleotide metabolism: a mutation in the adenylate kinase gene renders Yersinia pestis avirulent.

    PubMed Central

    Munier-Lehmann, Hélène; Chenal-Francisque, Viviane; Ionescu, Mihaela; Chrisova, Petya; Foulon, Jeannine; Carniel, Elisabeth; Bârzu, Octavian

    2003-01-01

    Nucleoside monophosphate kinases (NMPKs) are essential catalysts for bacterial growth and multiplication. These enzymes display high primary sequence identities among members of the family Enterobacteriaceae. Yersinia pestis, the causative agent of plague, belongs to this family. However, it was previously shown that its thymidylate kinase (TMPKyp) exhibits biochemical properties significantly different from those of its Escherichia coli counterpart [Chenal-Francisque, Tourneux, Carniel, Christova, Li de la Sierra, Barzu and Gilles (1999) Eur. J. Biochem. 265, 112-119]. In this work, the adenylate kinase (AK) of Y. pestis (AKyp) was characterized. As with TMPKyp, AKyp displayed a lower thermodynamic stability than other studied AKs. Two mutations in AK (Ser129Phe and Pro87Ser), previously shown to induce a thermosensitive growth defect in E. coli, were introduced into AKyp. The recombinant variants had a lower stability than wild-type AKyp and a higher susceptibility to proteolytic digestion. When the Pro87Ser substitution was introduced into the chromosomal adk gene of Y. pestis, growth of the mutant strain was altered at the non-permissive temperature of 37 degree C. In virulence testings, less than 50 colony forming units (CFU) of wild-type Y. pestis killed 100% of the mice upon subcutaneous infection, whereas bacterial loads as high as 1.5 x 10(4) CFU of the adk mutant were unable to kill any animals. PMID:12879903

  19. The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity.

    PubMed

    Fulcher, Nanette B; Holliday, Phillip M; Klem, Erich; Cann, Martin J; Wolfgang, Matthew C

    2010-05-01

    Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signalling molecule adenosine 3', 5'-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems.

  20. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    PubMed

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

  1. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia*

    PubMed Central

    Dong, Qian; Ernst, Sarah E.; Ostedgaard, Lynda S.; Shah, Viral S.; Ver Heul, Amanda R.; Welsh, Michael J.; Randak, Christoph O.

    2015-01-01

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

  2. Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

    PubMed

    Sorrentino, Luca; Calogero, Alessandra Maria; Pandini, Vittorio; Vanoni, Maria Antonietta; Sevrioukova, Irina F; Aliverti, Alessandro

    2015-12-01

    Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide.

  3. Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents.

    PubMed

    Matarlo, Joe S; Evans, Christopher E; Sharma, Indrajeet; Lavaud, Lubens J; Ngo, Stephen C; Shek, Roger; Rajashankar, Kanagalaghatta R; French, Jarrod B; Tan, Derek S; Tonge, Peter J

    2015-10-27

    MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.

  4. Mechanism of MenE Inhibition by Acyl-Adenylate Analogues and Discovery of Novel Antibacterial Agents

    PubMed Central

    Sharma, Indrajeet; Lavaud, Lubens J.; Ngo, Stephen C.; Shek, Roger; Rajashankar, Kanagalaghatta R.; French, Jarrod B.; Tan, Derek S.; Tonge, Peter J.

    2015-01-01

    MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1) which has an IC50 value of ≤ 25 nM for the Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in S. aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ~1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure–activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto-acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively-charged keto-acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future. PMID:26394156

  5. Antagonism of histamine-activated adenylate cyclase in brain by D-lysergic acid diethylamide.

    PubMed

    Green, J P; Johnson, C L; Weinstein, H; Maayani, S

    1977-12-01

    D-Lysergic acid diethylamide and D-2-bromolysergic acid diethylamide are competitive antagonists of the histamine activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); E.C. 4.6.1.1] in broken cell preparations of the hippocampus and cortex of guinea pig brain. The adenylate cyclase is linked to the histamine H2-receptor. Both D-lysergic acid diethylamide and D-2-bromolysergic acid diethylamide show topological congruency with potent H2-antagonists. D-2-Bromolysergic acid diethylamide is 10 times more potent as an H2-antagonist than cimetidine, which has been the most potent H2-antagonist reported, and D-lysergic acid diethylamide is about equipotent to cimetidine. Blockade of H2-receptors could contribute to the behavioral effects of D-2-bromolysergic acid diethylamide and D-lysergic acid diethylamide.

  6. Antagonism of histamine-activated adenylate cyclase in brain by D-lysergic acid diethylamide.

    PubMed Central

    Green, J P; Johnson, C L; Weinstein, H; Maayani, S

    1977-01-01

    D-Lysergic acid diethylamide and D-2-bromolysergic acid diethylamide are competitive antagonists of the histamine activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); E.C. 4.6.1.1] in broken cell preparations of the hippocampus and cortex of guinea pig brain. The adenylate cyclase is linked to the histamine H2-receptor. Both D-lysergic acid diethylamide and D-2-bromolysergic acid diethylamide show topological congruency with potent H2-antagonists. D-2-Bromolysergic acid diethylamide is 10 times more potent as an H2-antagonist than cimetidine, which has been the most potent H2-antagonist reported, and D-lysergic acid diethylamide is about equipotent to cimetidine. Blockade of H2-receptors could contribute to the behavioral effects of D-2-bromolysergic acid diethylamide and D-lysergic acid diethylamide. Images PMID:23536

  7. Assessment of industrial sewage impacts by adenylate energy charge measurements in the bivalve Cerastoderma edule.

    PubMed

    Picado, A M; Le Gal, Y

    1990-02-01

    Laboratory toxicity tests performed on the bivalve Cerastoderma edule submitted to sublethal concentrations of paper mill effluent revealed significant decreases of adenylate energy charge (AEC), and changes in the total adenylate pool were observed in a 24-hr period even for the lowest concentration of pollutant tested. Field transfer experiments of C. edule from a safe zone to polluted areas of the Sado estuary were carried out at two different times of the year. Close proximity to sewage outfall was shown to result in significant decreases in AEC values within 24 hr. One week after transfer, either normal AEC values were found or the organisms died, depending on the location of the sampling station.

  8. Substrate specificity and biochemical characterization of an adenylation domain from an obligate marine actinomycete.

    PubMed

    Shu, Xiao; Ma, Yanling; Lu, Chong; Lei, Ming; Liu, Yanli; Liu, Ke; Xiong, Guomei; Xia, Sisi; Zhao, Zhijuan; Luo, Wenkai; Fu, Qiao; Qi, Chao

    2015-05-01

    Salinispora arenicola CNS-205 was a first-isolated obligate marine actinomycete. A gene (sare0357), annotated as ''amino acid adenylation domain'' located on the genome of Salinispora arenicola CNS-205, was cloned and characterized. The recombinant target protein Sare0357 was expressed in E. coli. Sare0357 specifically recognized and activated tryptophan (Trp) and phenylalanine (Phe). The basic kinetic parameters of Sare0357 for Trp were K m = 0.04 mM, V max = 2.1 μM/min, k cat = 14.2 min(-1), and for Phe were K m = 0.03 mM, V max = 1.6 μM/min, kcat = 10.4 min(-1). Our data elucidated Sare0357 biological role and biochemical properties as a Trp and Phe-activating adenylation domain.

  9. Inhibition of denuded mouse oocyte meiotic maturation by forskolin, an activator of adenylate cyclase.

    PubMed

    Urner, F; Herrmann, W L; Baulieu, E E; Schorderet-Slatkine, S

    1983-09-01

    Forskolin, a diterpene that activates rapidly adenylate cyclase activity in several somatic cell systems, prevents spontaneous meiotic maturation of denuded mouse oocytes (ED50 of inhibition approximately 2.5 microM), unlike cholera toxin. The oocyte is sensitive to the action of forskolin during the period preceding germinal vesicle breakdown (GVBD). Washing of the cells abolishes the effect. The diterpene potentiates the inhibitory effect of iso-butyl-methyl-xanthine (IBMX), a phosphodiesterase inhibitor, and it increases cAMP concentration in the oocytes. These findings not only confirm the antagonistic effect of cAMP on the first step of meiosis reinitiation (GVBD) in mammalian oocytes, but also provide the first demonstration of a functional adenylate cyclase system in mammalian oocytes upon which regulatory signals may act.

  10. Direct interaction between the catalytic subunit of the calmodulin-sensitive adenylate cyclase from bovine brain with /sup 125/I-labeled wheat germ agglutinin and /sup 125/I-labeled calmodulin

    SciTech Connect

    Minocherhomjee, A.M.; Selfe, S.; Flowers, N.J.; Storm, D.R.

    1987-07-14

    A calmodulin-sensitive adenylate cyclase has been purified to apparent homogeneity from bovine cerebral cortex using calmodulin-Sepharose followed by forskolin-Sepharose and wheat germ agglutinin-Sepharose. The final product appeared as one major polypeptide of approximately 135,000 daltons on sodium dodecyl sulfate-polyacrylamide gels. This polypeptide was a major component of the protein purified through calmodulin-Sepharose. The catalytic subunit was stimulated 3-4-fold by calmodulin (CaM) with a turnover number greater than 1000 min/sup -1/ and was directly inhibited by adenosine. The catalytic subunit of the enzyme interacted directly with /sup 125/I-CaM on a sodium dodecyl sulfate-polyacrylamide gel overlay system, and this interaction was Ca/sup 2 +/ concentration dependent. In addition, the catalytic subunit was shown to directly bind /sup 125/I-labeled wheat germ agglutinin using a sodium dodecyl sulfate-polyacrylamide gel overlay technique, and N-acetylglucosamine inhibited binding of the lectin to the catalytic subunit. Calmodulin did not inhibit binding of wheat germ agglutinin to the catalytic subunit, and the binding of calmodulin was unaffected by wheat germ agglutinin. These data illustrate that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is a glycoprotein which interacts directly with calmodulin and that adenosine can inhibit the enzyme without intervening receptors or G coupling proteins. It is concluded that the catalytic subunit of adenylate cyclase is a transmembrane protein with a domain accessible from the outer surface of the cell.

  11. Two-dimensional NMR studies of the porcine muscle adenylate kinase

    SciTech Connect

    Klaus, W.; Scharf, M.; Zimmermann, S.; Roesch, P.

    1988-07-26

    Porcine adenylate kinase was subjected to one- and two-dimensional proton NMR studies in order to identify amino acid spin systems and obtain sequence-specific resonance assignments. With a combination of results from a map of side-chain distances resulting from the refined X-ray crystallographic data and nuclear Overhauser effect spectroscopy (NOESY), assignments are suggested for all the aromatic spin systems.

  12. Dysregulation of Alternative Poly-adenylation as a Potential Player in Autism Spectrum Disorder

    PubMed Central

    Szkop, Krzysztof J.; Cooke, Peter I. C.; Humphries, Joanne A.; Kalna, Viktoria; Moss, David S.; Schuster, Eugene F.; Nobeli, Irene

    2017-01-01

    We present here the hypothesis that alternative poly-adenylation (APA) is dysregulated in the brains of individuals affected by Autism Spectrum Disorder (ASD), due to disruptions in the calcium signaling networks. APA, the process of selecting different poly-adenylation sites on the same gene, yielding transcripts with different-length 3′ untranslated regions (UTRs), has been documented in different tissues, stages of development and pathologic conditions. Differential use of poly-adenylation sites has been shown to regulate the function, stability, localization and translation efficiency of target RNAs. However, the role of APA remains rather unexplored in neurodevelopmental conditions. In the human brain, where transcripts have the longest 3′ UTRs and are thus likely to be under more complex post-transcriptional regulation, erratic APA could be particularly detrimental. In the context of ASD, a condition that affects individuals in markedly different ways and whose symptoms exhibit a spectrum of severity, APA dysregulation could be amplified or dampened depending on the individual and the extent of the effect on specific genes would likely vary with genetic and environmental factors. If this hypothesis is correct, dysregulated APA events might be responsible for certain aspects of the phenotypes associated with ASD. Evidence supporting our hypothesis is derived from standard RNA-seq transcriptomic data but we suggest that future experiments should focus on techniques that probe the actual poly-adenylation site (3′ sequencing). To address issues arising from the use of post-mortem tissue and low numbers of heterogeneous samples affected by confounding factors (such as the age, gender and health of the individuals), carefully controlled in vitro systems will be required to model the effect of calcium signaling dysregulation in the ASD brain. PMID:28955198

  13. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    SciTech Connect

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  14. Stimulatory and inhibitory effects of forskolin on adenylate cyclase in rat normal hepatocytes and hepatoma cells.

    PubMed

    Miyamoto, K; Sanae, F; Koshiura, R; Matsunaga, T; Takagi, K; Satake, T; Hasegawa, T

    1989-02-01

    Forskolin synergistically potentiated adenosine 3',5'-cyclic monophosphate formation by prostaglandin E1 (PGE1) in rat normal hepatocytes freshly prepared by collagenase digestion and rat ascites hepatoma AH66 cells, but dose-dependently inhibited the accumulation by PGE1 in AH66F cells. Forskolin activated adenylate cyclase in a dose-dependent manner in homogenates of all cell lines. In normal hepatocytes and AH66 cells, simultaneous addition of forskolin and other adenylate cyclase activators [isoproterenol (IPN), PGE1, guanosine 5'-triphosphate sodium salt (GTP), 5'-guanylylimidodiphosphate sodium salt (Gpp (NH)p), NaF, cholera toxin, islet activating protein and MnCl2] gave greater than additive responses. On the other hand, in AH66F cells, the effect of forskolin on adenylate cyclase was hardly influenced by GTP, but forskolin diminished the activities induced by high concentrations of GTP to that by the diterpene alone. Forskolin also significantly inhibited the PGE1-stimulated and the guanine nucleotide binding regulatory protein-stimulated activities. Because AH66F cells were insensitive to IPN, the combination with forskolin and IPN gave similar activity to that obtained with the diterpene alone. The effect of forskolin on the activation by manganese ion was neither synergistic nor inhibitory but was additive in AH66F cells. These results suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide binding regulatory protein and the catalytic unit in normal hepatocytes and AH66 cells, but in AH66F cells forskolin interferes with the coupling of the two components of adenylate cyclase.

  15. Adenylate cyclase and the search for new compounds with the clinical profile of lithium.

    PubMed

    Belmaker, R H

    1984-01-01

    It is possible to evaluate the beta-adrenergic receptor-adenylate cyclase complex in the human periphery by measuring the plasma cyclic AMP rise after adrenergic agonists. A clinical trial of the beta 2 adrenergic agonist salbutamol in depression provided an opportunity to test whether adrenergic receptor subsensitivity does occur during clinical antidepressant treatment. After 1 and 3 weeks of oral salbutamol treatment, depression scores declined significantly in 11 depressed patients, while the plasma cyclic AMP response to i.v. salbutamol declined over 60%. The results support the concept that receptor sensitivity changes occur during human antidepressant therapy. Data are presented that Li, too, markedly reduces activity of beta-adrenergic adenylate cyclase in humans. The effect was evaluated by studying the effect of Li at therapeutic serum concentrations on the plasma cyclic AMP response to subcutaneous epinephrine. The Li effect is specific, since the plasma cyclic AMP response to glucagon is not inhibited. In rat cortical slices Li inhibition of noradrenaline-induced cyclic AMP accumulation is clearly demonstrable only at concentrations close to 2 mM Li. However, fresh human brain slices from edges of surgically-removed tumors show Li inhibition at 1 mM Li concentrations. These results imply that in brain as well as periphery, human noradrenergic adenylate cyclase is inhibited by therapeutic concentrations of Li. Demeclocyclin, a tetracycline-derived antibiotic, was found to inhibit noradrenaline-sensitive adenylate cyclase in rat cortical slices and to inhibit amphetamine-induced hyperactivity in rats in an open field. Clinical trials should search for new compounds with the clinical profile of Li.

  16. Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

    PubMed Central

    Eggerickx, D; Denef, J F; Labbe, O; Hayashi, Y; Refetoff, S; Vassart, G; Parmentier, M; Libert, F

    1995-01-01

    A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed. Images Figure 4 Figure 5 PMID:7639700

  17. Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform

    PubMed Central

    Cámara, María de los Milagros; Bouvier, León A.; Canepa, Gaspar E.; Miranda, Mariana R.; Pereira, Claudio A.

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N-terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 3′ UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes. PMID:23409202

  18. Relationship between muscarinic receptor occupancy and adenylate cyclase inhibition in the rabbit myocardium

    SciTech Connect

    Ehlert, F.J.

    1985-11-01

    The muscarinic receptor-binding properties of a series of muscarinic drugs were compared with their effects on adenylate cyclase in membranes of the rabbit myocardium. When measured by competitive inhibition of (TH)-N-methylscopolamine binding, the competition curves of the various agonists were adequately described by the ternary complex model. This model assumes that the receptor can bind reversibly with a guanine nucleotide binding protein in the membrane and that the affinity of the agonist for the receptor-guanine nucleotide-binding protein complex is higher than that for the free receptor. A satisfactory fit of the ternary complex model to the data could only be achieved assuming that very little receptor is precoupled with the guanine nucleotide-binding protein in the absence of agonist. There was good agreement between the efficacy of each agonist as measured by inhibition of adenylate cyclase and the estimate of the positive cooperativity between the binding of the agonist receptor complex and the guanine nucleotide-binding protein. Guanosine 5'-triphosphate (0.1 mM) had no significant effect on the binding of (TH)N-methylscopolamine but caused an increase in the concentration of the various agonists required for half-maximal receptor occupancy. There was good correlation between efficacy as measured by inhibition of adenylate cyclase and the influence of guanosine 5'-triphosphate on binding properties.

  19. Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover.

    PubMed Central

    Stein, R; Pinkas-Kramarski, R; Sokolovsky, M

    1988-01-01

    The rat M1 muscarinic receptor gene was cloned and expressed in a rat cell line lacking endogenous muscarinic receptors. Assignment of the cloned receptors to the M1 class was pharmacologically confirmed by their high affinity for the M1-selective muscarinic antagonist pirenzepine and low affinity for the M2-selective antagonist AF-DX-116. Guanylyl imidodiphosphate [Gpp(NH)p] converted agonist binding sites on the receptor, from high-affinity to the low-affinity state, thus indicating that the cloned receptors couple to endogenous G-proteins. The cloned receptors mediated both adenylate cyclase inhibition and phosphoinositide hydrolysis, but by different mechanisms. Pertussis toxin blocked the inhibition of adenylate cyclase (indicating coupling of the receptor to inhibitory G-protein), but did not affect phosphoinositide turnover. Furthermore, the stimulation of phosphoinositide hydrolysis was less efficient than the inhibition of adenylate cyclase. These findings demonstrate that cloned M1 receptors are capable of mediating multiple responses in the cell by coupling to different effectors, possibly to different G-proteins. Images PMID:2846274

  20. Molecular and functional characterization of a Trypanosoma cruzi nuclear adenylate kinase isoform.

    PubMed

    Cámara, María de los Milagros; Bouvier, León A; Canepa, Gaspar E; Miranda, Mariana R; Pereira, Claudio A

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N-terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 3' UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes.

  1. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    PubMed Central

    Israeli, Ma’ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  2. Persistent stimulation of adenylate cyclase and urea transport by an AVP photolabel

    SciTech Connect

    Eggena, P.; Ma, C.L.; Fahrenholz, F.; Schwartz, I.L.

    1985-07-01

    The effects of a photoaffinity label for arginine vasopressin receptors, (Phe2, Phe(p-N3)3)AVP (N3-AVP), on urea permeability and adenylate cyclase activity have been investigated in the toad urinary bladder. This compound, when activated by ultraviolet light, induced a maximal and persistent increase in the urea permeability of the intact bladder and a persistent increase in the adenylate cyclase activity of toad bladder epithelial cell homogenates. Covalent attachment of the analogue to target tissue during photolysis was equivalent at 4 and 20 degrees C. Bladders exposed to N3-AVP in the presence of AVP during photolysis were substantially less permeable to urea than controls that had been exposed to N3-AVP alone. These findings constitute further evidence in support of the previous suggestion that N3-AVP binds covalently to AVP receptors and, in addition, demonstrates that N3-AVP evokes a persistent increase in adenylate cyclase activity which, in turn, triggers a persistent increase in bladder permeability to urea.

  3. The intact CFTR protein mediates ATPase rather than adenylate kinase activity.

    PubMed

    Ramjeesingh, Mohabir; Ugwu, Francisca; Stratford, Fiona L L; Huan, Ling-Jun; Li, Canhui; Bear, Christine E

    2008-06-01

    The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

  4. Multiforms of mammalian adenylate kinase and its monoclonal antibody against AK1.

    PubMed

    Kurokawa, Y; Takenaka, H; Sumida, M; Oka, K; Hamada, M; Kuby, S A

    1990-01-01

    An attempt has been made to determine the intracellular distribution of the multiforms of the adenylate kinase (AK) isoenzymes in mammalian tissues, to shed some light on their physiological roles, especially in energy metabolism. The adenylate kinase zymograms obtained from isoelectric focusing yielded two typical isoform patterns: (1) with a pI greater than or equal to 9 and 8.6, specific for bovine skeletal muscle, heart, aorta and brain, and (2) with a pI = 7.9 and 7.1, specific for liver and kidney. Pattern (1) was attributed to the cytosolic isoenzyme (AK1) as demonstrated by immunostaining with anti-AK1. Pattern (2) was attributed to the mitochondrial isoenzyme (AK2). These results were largely confirmed by chromatofocusing experiments. The AK1 isoenzyme was partially purified from the cytosol fraction of bovine aortic smooth muscle and had an apparent Mr of 23.5 kilodaltons. Its kinetic features are discussed from a comparative standpoint. Finally, the human serum AK1 isoform was also detected by Western blotting with a monoclonal antibody directed against crystalline porcine muscle AK1. These results are to form the basis of further studies on the 'aberrant' adenylate kinase isoenzyme from the serum of Duchenne muscular dystrophics.

  5. Evidence Against the Presence of 3′, 5′-Cyclic Adenosine Monophosphate and Relevant Enzymes in Lactobacillus plantarum

    PubMed Central

    Sahyoun, N.; Durr, I. F.

    1972-01-01

    Analysis of cells of Lactobacillus plantarum, starved or undergoing induction, showed no 3′, 5′-cyclic adenosine monophosphate (cAMP). Neither adenyl cyclase nor 3′, 5′-cAMP phosphodiesterase was detected in extracts. Extracts of L. plantarum did not inhibit these two enzymes of Escherichia coli K-12, strain W1435. Incubation of adenosine triphosphate (ATP)-U-14C with cells or various cell-free fractions of L. plantarum did not produce labeled 3′, 5′-cAMP. Of various 3′, 5′-cyclic and acyclic nucleotides tested, only 3′, 5′-cAMP, ATP, and yeast adenylic acid stimulated l-arabinose isomerase. Yeast adenylic acid was two to four times as effective as 3′, 5′-cAMP or ATP. 2′, 3′-cAMP was not effective. PMID:4342815

  6. Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy.

    PubMed

    Fry, D C; Byler, D M; Susi, H; Brown, E M; Kuby, S A; Mildvan, A S

    1988-05-17

    The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and

  7. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  8. Adenylate kinase release as a high-throughput-screening-compatible reporter of bacterial lysis for identification of antibacterial agents.

    PubMed

    Jacobs, Anna C; Didone, Louis; Jobson, Jennielle; Sofia, Madeline K; Krysan, Damian; Dunman, Paul M

    2013-01-01

    Adenylate kinase (AK) is a ubiquitous intracellular enzyme that is released into the extracellular space upon cell lysis. We have shown that AK release serves as a useful reporter of bactericidal agent activity and can be exploited for antimicrobial screening purposes. The AK assay exhibits improved sensitivity over that of growth-based assays and can detect agents that are active against bacteria in clinically relevant growth states that are difficult to screen using conventional approaches, such as small colony variants (SCV) and bacteria within established biofilms. The usefulness of the AK assay was validated by screening a library of off-patent drugs for agents that exhibit antimicrobial properties toward a variety of bacterial species, including Escherichia coli and all members of the "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The assay detected antibiotics within the library that were expected to be active against the organism screened. Moreover, 38 drugs with no previously reported antibacterial activity elicited AK release. Four of these were acquired, and all were verified to exhibit antimicrobial activity by standard susceptibility testing. Two of these molecules were further characterized. The antihistamine, terfenadine, was active against S. aureus planktonic, SCV population, and biofilm-associated cells. Tamoxifen, an estrogen receptor antagonist, was active toward E. faecium in vitro and also reduced E. faecium pathogenesis in a Galleria mellonella infection model. Our data demonstrate that the AK assay provides an attractive screening approach for identifying new antimicrobial agents. Further, terfenadine and tamoxifen may represent novel antimicrobial drug development scaffolds.

  9. Adenylate Kinase Release as a High-Throughput-Screening-Compatible Reporter of Bacterial Lysis for Identification of Antibacterial Agents

    PubMed Central

    Jacobs, Anna C.; DiDone, Louis; Jobson, Jennielle; Sofia, Madeline K.

    2013-01-01

    Adenylate kinase (AK) is a ubiquitous intracellular enzyme that is released into the extracellular space upon cell lysis. We have shown that AK release serves as a useful reporter of bactericidal agent activity and can be exploited for antimicrobial screening purposes. The AK assay exhibits improved sensitivity over that of growth-based assays and can detect agents that are active against bacteria in clinically relevant growth states that are difficult to screen using conventional approaches, such as small colony variants (SCV) and bacteria within established biofilms. The usefulness of the AK assay was validated by screening a library of off-patent drugs for agents that exhibit antimicrobial properties toward a variety of bacterial species, including Escherichia coli and all members of the “ESKAPE” pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The assay detected antibiotics within the library that were expected to be active against the organism screened. Moreover, 38 drugs with no previously reported antibacterial activity elicited AK release. Four of these were acquired, and all were verified to exhibit antimicrobial activity by standard susceptibility testing. Two of these molecules were further characterized. The antihistamine, terfenadine, was active against S. aureus planktonic, SCV population, and biofilm-associated cells. Tamoxifen, an estrogen receptor antagonist, was active toward E. faecium in vitro and also reduced E. faecium pathogenesis in a Galleria mellonella infection model. Our data demonstrate that the AK assay provides an attractive screening approach for identifying new antimicrobial agents. Further, terfenadine and tamoxifen may represent novel antimicrobial drug development scaffolds. PMID:23027196

  10. Role of phosphodiesterase and adenylate cyclase isozymes in murine colonic glucagon-like peptide 1 secreting cells

    PubMed Central

    Friedlander, Ronn S; Moss, Catherine E; Mace, Jessica; Parker, Helen E; Tolhurst, Gwen; Habib, Abdella M; Wachten, Sebastian; Cooper, Dermot M; Gribble, Fiona M; Reimann, Frank

    2011-01-01

    BACKGROUND AND PURPOSE Glucagon-like peptide-1 (GLP-1) is secreted from enteroendocrine L-cells after food intake. Increasing GLP-1 signalling either through inhibition of the GLP-1 degrading enzyme dipeptidyl-peptidase IV or injection of GLP-1-mimetics has recently been successfully introduced for the treatment of type 2 diabetes. Boosting secretion from the L-cell has so far not been exploited, due to our incomplete understanding of L-cell physiology. Elevation of cyclic adenosine monophosphate (cAMP) has been shown to be a strong stimulus for GLP-1 secretion and here we investigate the activities of adenylate cyclase (AC) and phosphodiesterase (PDE) isozymes likely to shape cAMP responses in L-cells. EXPERIMENTAL APPROACH Expression of AC and PDE isoforms was quantified by RT-PCR. Single cell responses to stimulation or inhibition of AC and PDE isoforms were monitored with real-time cAMP probes. GLP-1 secretion was assessed by elisa. KEY RESULTS Quantitative PCR identified expression of protein kinase C- and Ca2+-activated ACs, corresponding with phorbolester and cytosolic Ca2+-stimulated cAMP elevation. Inhibition of PDE2, 3 and 4 were found to stimulate GLP-1 secretion from murine L-cells in primary culture. This corresponded with cAMP elevations monitored with a plasma membrane targeted cAMP probe. Inhibition of PDE3 but not PDE2 was further shown to prevent GLP-1 secretion in response to guanylin, a peptide secreted into the gut lumen, which had not previously been implicated in L-cell secretion. CONCLUSIONS AND IMPLICATIONS Our results reveal several mechanisms shaping cAMP responses in GLP-1 secreting cells, with some of the molecular components specifically expressed in L-cells when compared with their epithelial neighbours, thus opening new strategies for targeting these cells therapeutically. PMID:21054345

  11. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    SciTech Connect

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  12. Cellular levels of feedback regulator of adenylate cyclase and the effect of epinephrine and insulin.

    PubMed Central

    Ho, R j; Russell, T R; Asakawa, T; Sutherland, E W

    1975-01-01

    We have obtained direct evidence that shows the cellular formation and subsequent release of a potent inhibitor (feedback regulator) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by adipocytes, upon stimulation with epinephrine. The appearance of such a feedback regulator in adipocytes preceded its release into the medium. During a 30 min incubation, intracellular regulator levels rose rapidly and reached 39-61 units/g of adipocyte at 10 min. Release of inhibitor into the medium increased slowly and was 11-16 units/g of adipocyte at 10 min. Upon continued incubation, the cells at 30 min contained 30-41 units/g of ingibitor, slightly less than the content at 30 min; meanwhile, the medium content rose more than 3-fold. The inhibitor from both locations appeared to have the same characteristics, judging from the purification procedures and the biological activities on hormone-stimulated adenylate cyclase. Adenylate cyclase was inhibited by the feedback regulator in vitro when either epinephrine, corticotropin (ACTH), or glucagon was used as activator. The site of action of this inhibitor is therefore most likely beyond the specific hormone receptors. A new in vitro action of insulin has been found. Insulin, 50-500 microunits/ml, inhibited the formation and release of this factor from isolated rat or hamster adipocytes by 29-81% after these cells were stimulated by hormones that raise intracellular adenosine 3':5'-cyclic monophosphate. This factor enhaced the effect of insulin in lowering the adenosine 3':5'-cyclic monophosphate levels in fresh rat adipocytes. A reduced formation of such a factor may modify the metabolic events in adipocytes, and some as yet unexplained effects of insulin could therefore be linked to the metabolic effects of this factor. PMID:174073

  13. On the dynamics of the adenylate energy system: homeorhesis vs homeostasis.

    PubMed

    De la Fuente, Ildefonso M; Cortés, Jesús M; Valero, Edelmira; Desroches, Mathieu; Rodrigues, Serafim; Malaina, Iker; Martínez, Luis

    2014-01-01

    Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for

  14. On the Dynamics of the Adenylate Energy System: Homeorhesis vs Homeostasis

    PubMed Central

    De la Fuente, Ildefonso M.; Cortés, Jesús M.; Valero, Edelmira; Desroches, Mathieu; Rodrigues, Serafim; Malaina, Iker; Martínez, Luis

    2014-01-01

    Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for

  15. Crystal Structures of the Adenylate Sensor from Fission Yeast AMP-Activated Protein Kinase

    SciTech Connect

    Townley,R.; Shapiro, L.

    2007-01-01

    The 5'-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular adenosine triphosphate (ATP) and AMP levels. Here we report crystal structures at 2.6 and 2.9 Angstrom resolution for ATP- and AMP-bound forms of a core {alpha}{beta}{gamma} adenylate-binding domain from the fission yeast AMPK homologue. ATP and AMP bind competitively to a single site in the {gamma} subunit, with their respective phosphate groups positioned near function-impairing mutants. Surprisingly, ATP binds without counter ions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.

  16. Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases.

    PubMed

    Hara, Ryotaro; Suzuki, Ryohei; Kino, Kuniki

    2015-05-15

    We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp-hydroxamate-Fe(3+) complex and the formation of l-Trp-l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.

  17. Role of protein kinase C on the acute desensitization of renal cortical adenylate cyclase to parathyroid hormone.

    PubMed

    Bellorin-Font, E; López, C; Díaz, K; Pernalete, N; López, M; Starosta, R

    1995-01-01

    The mechanisms of adenylate cyclase desensitization to parathyroid hormone are still unclear. Current evidence suggest that the signal generated after PTH binding to receptors results in activation of adenylate cyclase and stimulation of phospholipase C with subsequent activation of protein kinase C. Recent studies have suggested a role of protein kinase C on the regulation of the PTH-dependent receptor-adenylate cyclase system in cultured cells. Therefore, the present studies were conducted to examine the role of protein kinase C on the desensitization of canine renal cortical adenylate cyclase after an acute exposure in vivo to PTH. A group of normal dogs were treated with a single intravenous injection of 1 microgram/k of syn bPTH (1-34) or Nle bPTH (3-34). Ten minutes later, animals were subjected to bilateral nephrectomy and the kidney cortex processed for preparations of basolateral membranes for determinations of adenylate cyclase activity, as well as membrane and cytosolic fractions for analysis of protein kinase C activity. Animals not treated with PTH were used as controls. PTH administration in vivo resulted in a 46.9 +/- 9.3% decrease in maximal adenylate cyclase activity in vitro in response to syn bPTH (1-34) (P < 0.001). Likewise, PTH binding as measured with 125I-Nle8,18,Tyr34-bPTH (1-34)NH2 showed a 40 +/- 3% decrease. This alterations were associated with a marked translocation of protein kinase C from the cytosol to the membrane. Thus, protein kinase C activity in membrane fractions increased from 160.6 +/- 44.8 pmol Pi/min in controls to 500.4 +/- 123 in PTH treated dogs (P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Multiple effects of phorbol esters on hormone-sensitive adenylate cyclase activity in S49 lymphoma cells

    SciTech Connect

    Bell, J.D.; Brunton, L.L.

    1987-06-01

    In S49 lymphoma cells, 12-O-tetradecanoyl phorbol-13-acetate (TPA) enhances adenylate cyclase activity and doubles cAMP accumulation in response to ..beta..-adrenergic stimulation at 37/sup 0/C, putatively via the action of protein kinase C. at 27/sup 0/C, TPA has the opposite effect, inhibiting cAMP production in response to isoproterenol by approx. 25%. TPA also inhibits the response to prostaglandin E/sub 1/ (PGE/sub 1/), another stimulant of hormone-sensitive adenylate cyclase in these cells, by 30% at 37/sup 0/C and almost 50% at 27/sup 0/C. In contrast, TPA enhances responses to forskolin and cholera toxin at both 27 and 37/sup 0/C. In membranes from cells treated with TPA, PGE/sub 1/-stimulated adenylate cyclase activity is inhibited by 50%, whereas the catalytic activity stimulated by NaF or forskolin is enhanced. TPA reduces the potency of both PGE/sub 1/ and isoproterenol for cAMP generation by 50%. TPA causes a similar decrease in ..beta..-adrenergic agonist affinity with no reduction in the density of either antagonist of agonist binding sites in wild type cells and in cells lacking the ..cap alpha..-subunit of the stimulatory transducer protein (G/sub s/) (cyc/sup -/) or lacking functional receptor G/sub s/ coupling (UNC). Therefore, TPA has at least three functionally distinct effects on hormone-sensitive adenylate cyclase in S49 cells. The authors conclude that multiple and opposing effects of TPA on hormone-sensitive adenylate cyclase occur simultaneously within the same cell, affecting the responses to several agonists differently. In addition, the data offer a mechanism by which a cell can achieve heterogeneous efficacies to hormones that activate adenylate cyclase.

  19. New isozyme systems for maize (Zea mays L.): aconitate hydratase, adenylate kinase, NADH dehydrogenase, and shikimate dehydrogenase.

    PubMed

    Wendel, J F; Goodman, M M; Stuber, C W; Beckett, J B

    1988-06-01

    Electrophoretic variation and inheritance of four novel enzyme systems were studied in maize (Zea mays L.). A minimum of 10 genetic loci collectively encodes isozymes of aconitate hydratase (ACO; EC 4.2.1.3.), adenylate kinase (ADK; EC 2.7.4.3), NADH dehydrogenase (DIA; EC 1.6.99.-), and shikimate dehydrogenase (SAD; EC 1.1.1.25). At least four loci are responsible for the genetic control of ACO. Genetic data for two of the encoding loci, Aco1 and Aco4, demonstrated that at least two maize ACOs are active as monomers. Analysis of organellar preparations suggests that ACO1 and ACO4 are localized in the cytosolic and mitochondrial subcellular fractions, respectively. Maize ADK is encoded by a single nuclear locus, Adk1, governing monomeric enzymes that are located in the chloroplasts. Two cytosolic and two mitochondrial forms of DIA were electrophoretically resolved. Segregation analyses demonstrated that the two cytosolic isozymes are controlled by separate loci, Dia1 and Dia2, coding for products that are functional as monomers (DIA1) and dimers (DIA2). The major isozyme of SAD is apparently cytosolic, although an additional faintly staining plastid form may be present. Alleles at Sad1 are each associated with two bands that cosegregate in controlled crosses. Linkage analyses and crosses with B-A translocation stocks were effective in determining the map locations of six loci, including the previously described but unmapped locus Acp4. Several of these loci were localized to sparsely mapped regions of the genome. Dia2 and Acp4 were placed on the distal portion of the long arm of chromosome 1, 12.6 map units apart. Dia1 was localized to chromosome 2, 22.2 centimorgans (cM) from B1. Aco1 was mapped to chromosome 4, 6.2 cM from su1. Adk1 was placed on the poorly marked short arm of chromosome 6, 8.1 map units from rgd1. Less than 1% recombination was observed between Glu1 (on chromosome 10) and Sad1. In contrast to many other maize isozyme systems, there was little

  20. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  1. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  2. Adenylate cyclase toxin (ACT) from Bordetella hinzii: characterization and differences from ACT of Bordetella pertussis.

    PubMed

    Donato, Gina M; Hsia, Hung-Lun J; Green, Candace S; Hewlett, Erik L

    2005-11-01

    Bordetella hinzii is a commensal respiratory microorganism in poultry but is increasingly being recognized as an opportunistic pathogen in immunocompromised humans. Although associated with a variety of disease states, practically nothing is known about the mechanisms employed by this bacterium. In this study, we show by DNA sequencing and reverse transcription-PCR that both commensal and clinical strains of B. hinzii possess and transcriptionally express cyaA, the gene encoding adenylate cyclase toxin (ACT) in other pathogenic Bordetella species. By Western blotting, we also found that B. hinzii produces full-length ACT protein in quantities that are comparable to those made by B. pertussis. In contrast to B. pertussis ACT, however, ACT from B. hinzii is less extractable from whole bacteria, nonhemolytic, has a 50-fold reduction in adenylate cyclase activity, and is unable to elevate cyclic AMP levels in host macrophages (nontoxic). The decrease in enzymatic activity is attributable, at least in part, to a decreased binding affinity of B. hinzii ACT for calmodulin, the eukaryotic activator of B. pertussis ACT. In addition, we demonstrate that the lack of intoxication by B. hinzii ACT may be due to the absence of expression of cyaC, the gene encoding the accessory protein required for the acylation of B. pertussis ACT. These results demonstrate the expression of ACT by B. hinzii and represent the first characterization of a potential virulence factor of this organism.

  3. Adenylate Cyclase AcyA Regulates Development, Aflatoxin Biosynthesis and Fungal Virulence in Aspergillus flavus.

    PubMed

    Yang, Kunlong; Qin, Qiuping; Liu, Yinghang; Zhang, Limei; Liang, Linlin; Lan, Huahui; Chen, Chihao; You, Yunchao; Zhang, Feng; Wang, Shihua

    2016-01-01

    Aspergillus flavus is one of the most important opportunistic pathogens of crops and animals. The carcinogenic mycotoxin, aflatoxins produced by this pathogen cause a health problem to human and animals. Since cyclic AMP signaling controls a range of physiological processes, like fungal development and infection when responding to extracellular stimuli in fungal pathogens, in this study, we investigated the function of adenylate cyclase, a core component of cAMP signaling, in aflatoxins biosynthesis and virulence on plant seeds in A. flavus. A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase. Severe defects in fungal growth, sporulation and sclerotia formation were observed in the acyA deletion mutant. The defect in radical growth could be partially rescued by exogenous cAMP analog. The acyA mutant was also significantly reduced in aflatoxins production and virulence. Similar to the former studies in other fungi, The acyA mutant showed enhancing tolerance to oxidative stress, but more sensitive to heat stress. Overall, the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development, aflatoxins biosynthesis and plant seed invasion in A. flavus.

  4. Adenylate Cyclase AcyA Regulates Development, Aflatoxin Biosynthesis and Fungal Virulence in Aspergillus flavus

    PubMed Central

    Yang, Kunlong; Qin, Qiuping; Liu, Yinghang; Zhang, Limei; Liang, Linlin; Lan, Huahui; Chen, Chihao; You, Yunchao; Zhang, Feng; Wang, Shihua

    2016-01-01

    Aspergillus flavus is one of the most important opportunistic pathogens of crops and animals. The carcinogenic mycotoxin, aflatoxins produced by this pathogen cause a health problem to human and animals. Since cyclic AMP signaling controls a range of physiological processes, like fungal development and infection when responding to extracellular stimuli in fungal pathogens, in this study, we investigated the function of adenylate cyclase, a core component of cAMP signaling, in aflatoxins biosynthesis and virulence on plant seeds in A. flavus. A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase. Severe defects in fungal growth, sporulation and sclerotia formation were observed in the acyA deletion mutant. The defect in radical growth could be partially rescued by exogenous cAMP analog. The acyA mutant was also significantly reduced in aflatoxins production and virulence. Similar to the former studies in other fungi, The acyA mutant showed enhancing tolerance to oxidative stress, but more sensitive to heat stress. Overall, the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development, aflatoxins biosynthesis and plant seed invasion in A. flavus. PMID:28066725

  5. Increase in the amount of adenylate cyclase in rat gastrocnemius muscle after denervation

    SciTech Connect

    Hashimoto, K.; Watanabe, Y.; Uchida, S.; Yoshida, H.

    1989-01-01

    After section of the sciatic nerve, the basal adenylate cyclase (AC) activity in rat gastrocnemius muscle increased 6-7 times per membrane protein and about 2 times per whole muscle in the following 30 or 40 days. The AC activity in the muscle 30 days after denervation was increased about 4 times by folskolin. Calcitonin gene-related peptide (CGRP) also increased the adenylate cyclase activity in the denervated muscle. The binding of (/sup 3/H)-forskolin to cells isolated from gastrocnemius muscle was examined to determine the amount of AC molecules. Inhibition of (/sup 3/H)-forskolin binding by increasing amounts of unlabeled forskolin gave a sigmoid curve with a IC/sub 50/ value of 3/times/10/sup /minus/7/M. Results showed that the number of (/sup 3/H)-forskolin binding sites per cell was higher on the denervated side than on the control side, like the basal AC activity. The IC/sub 50/ values for inhibition by unlabeled forskolin of binding of (/sup 3/H)-forskolin were similar to muscles on the control and denervated sides. These results suggest that an increase in the AC activity induced by denervation was due to an increase in the numbers of AC molecules in the muscle.

  6. Opening mechanism of adenylate kinase can vary according to selected molecular dynamics force field.

    PubMed

    Unan, Hulya; Yildirim, Ahmet; Tekpinar, Mustafa

    2015-07-01

    Adenylate kinase is a widely used test case for many conformational transition studies. It performs a large conformational transition between closed and open conformations while performing its catalytic function. To understand conformational transition mechanism and impact of force field choice on E. Coli adenylate kinase, we performed all-atom explicit solvent classical molecular dynamics simulations starting from the closed conformation with four commonly used force fields, namely, Amber99, Charmm27, Gromos53a6, Opls-aa. We carried out 40 simulations, each one 200 ns. We analyzed completely 12 of them that show full conformational transition from the closed state to the open one. Our study shows that different force fields can have a bias toward different transition pathways. Transition time scales, frequency of conformational transitions, order of domain motions and free energy landscapes of each force field may also vary. In general, Amber99 and Charmm27 behave similarly while Gromos53a6 results have a resemblance to the Opls-aa force field results.

  7. Binding of (/sup 3/H)forskolin to solubilized preparations of adenylate cyclase

    SciTech Connect

    Nelson, C.A.; Seamon, K.B.

    1988-01-01

    The binding of (/sup 3/H)forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating (/sup 3/H)forskolin bound to protein from free (/sup 3/H)forskolin by rapid filtration. The K/sub d/ for (/sup 3/H)forskolin binding to solubilized proteins was 14 nM which was similar to that for (/sup 3/H)forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for (/sup 3/H)forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. (/sup 3/H)forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmolmg protein which increased to 94 fmolmg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on (/sup 3/H)forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmolmgmin which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmolmgmin which was not stimulated by GppNHp or forskolin

  8. Opening mechanism of adenylate kinase can vary according to selected molecular dynamics force field

    NASA Astrophysics Data System (ADS)

    Unan, Hulya; Yildirim, Ahmet; Tekpinar, Mustafa

    2015-07-01

    Adenylate kinase is a widely used test case for many conformational transition studies. It performs a large conformational transition between closed and open conformations while performing its catalytic function. To understand conformational transition mechanism and impact of force field choice on E. Coli adenylate kinase, we performed all-atom explicit solvent classical molecular dynamics simulations starting from the closed conformation with four commonly used force fields, namely, Amber99, Charmm27, Gromos53a6, Opls-aa. We carried out 40 simulations, each one 200 ns. We analyzed completely 12 of them that show full conformational transition from the closed state to the open one. Our study shows that different force fields can have a bias toward different transition pathways. Transition time scales, frequency of conformational transitions, order of domain motions and free energy landscapes of each force field may also vary. In general, Amber99 and Charmm27 behave similarly while Gromos53a6 results have a resemblance to the Opls-aa force field results.

  9. Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions.

    PubMed

    Balestri, Francesco; Giannecchini, Michela; Sgarrella, Francesco; Carta, Maria Caterina; Tozzi, Maria Grazia; Camici, Marcella

    2007-02-01

    The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.

  10. GSK3β Mediates Renal Response to Vasopressin by Modulating Adenylate Cyclase Activity

    PubMed Central

    Patel, Satish; Hao, ChuanMing; Woodgett, James; Harris, Raymond

    2010-01-01

    Glycogen synthase kinase 3β (GSK3β), a serine/threonine protein kinase, is a key target of drug discovery in several diseases, including diabetes and Alzheimer disease. Because lithium, a potent inhibitor of GSK3β, causes nephrogenic diabetes insipidus, GSK3β may play a crucial role in regulating water homeostasis. We developed renal collecting duct-specific GSK3β knockout mice to determine whether deletion of GSK3β affects arginine vasopressin-dependent renal water reabsorption. Although only mildly polyuric under normal conditions, knockout mice exhibited an impaired urinary concentrating ability in response to water deprivation or treatment with a vasopressin analogue. The knockout mice had reduced levels of mRNA, protein, and membrane localization of the vasopressin-responsive water channel aquaporin 2 compared with wild-type mice. The knockout mice also expressed lower levels of pS256-AQP2, a phosphorylated form crucial for membrane trafficking. Levels of cAMP, a major regulator of aquaporin 2 expression and trafficking, were also lower in the knockout mice. Both GSK3β gene deletion and pharmacologic inhibition of GSK3β reduced adenylate cyclase activity. In summary, GSK3β inactivation or deletion reduces aquaporin 2 expression by modulating adenylate cyclase activity and cAMP generation, thereby impairing responses to vasopressin in the renal collecting duct. PMID:20056751

  11. Supersensitivity of beta-adrenoceptor coupled adenylate cyclase in pulmonary tissue of the spontaneously hypertensive rat

    SciTech Connect

    Kamibayashi, C.; Ramanathan, S. )

    1989-01-01

    Basal adenylate cyclase activity was similar in plasma membranes prepared from the lungs of 12 week old spontaneously hypertensive rats (SHR) and normotensive Wister Kyoto rats (WKY). However, sensitivity to Gpp (NH)p, isoproterenol plus GTP or Gpp (NH)p was significantly greater in the SHR. Beta-receptor density measured by ({sup 3}H)DHA binding was unaltered. The dissociation constant, K{sub d}, revealed a significantly greater binding affinity of the radioligand in the SHR compared with the WKY. Activity of G{sub s} was assessed by complementing S49 cyc{sup {minus}} acceptor membranes with lung cholate extract. Basal activity of the reconstituted system was decreased 43% in the SHR. However, sensitivity to NaF, Gpp(NH)p, and isoproterenol plus Gpp(NH)p was significantly elevated. These data suggest that desensitization of the adenylate cyclase complex is not a generalized response to chronic hypertension. A tissue specific increase in sympathetic drive appears to be responsible for the lowered concentration of cardiac beta-adrenoceptors in the SHR. In contrast, both indirect and direct evidence indicate an enhanced functional sensitivity of pulmonary G{sub s} in the hypertensive rats.

  12. Structure of the adenylation domain of NAD(+)-dependent DNA ligase from Staphylococcus aureus.

    PubMed

    Han, Seungil; Chang, Jeanne S; Griffor, Matt

    2009-11-01

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3'-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD(+)-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD(+)-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD(+)-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  13. Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

    SciTech Connect

    Han, Seungil; Chang, Jeanne S.; Griffor, Matt; Pfizer

    2010-09-17

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  14. High Inorganic Triphosphatase Activities in Bacteria and Mammalian Cells: Identification of the Enzymes Involved

    PubMed Central

    Lakaye, Bernard; Servais, Anne-Catherine; Scholer, Georges; Fillet, Marianne; Elias, Benjamin; Derochette, Jean-Michel; Crommen, Jacques; Wins, Pierre; Bettendorff, Lucien

    2012-01-01

    Background We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. Methodology/Principal Findings Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. Conclusions and General Significance We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling. PMID:22984449

  15. Leucine-rich repeats and carboxyl terminus are required for interaction of yeast adenylate cyclase with RAS proteins.

    PubMed Central

    Suzuki, N; Choe, H R; Nishida, Y; Yamawaki-Kataoka, Y; Ohnishi, S; Tamaoki, T; Kataoka, T

    1990-01-01

    A Saccharomyces cerevisiae gene encoding adenylate cyclase has been analyzed by deletion and insertion mutagenesis to localize regions required for activation by the Sa. cerevisiae RAS2 protein. The NH2-terminal 657 amino acids were found to be dispensable for the activation. However, almost all 2-amino acid insertions in the middle 600 residues comprising leucine-rich repeats and deletions in the COOH-terminal 66 residues completely abolished activation by the RAS2 protein, whereas insertion mutations in the other regions generally had no effect. Chimeric adenylate cyclases were constructed by swapping the upstream and downstream portions surrounding the catalytic domains between the Sa. cerevisiae and Schizosaccharomyces pombe adenylate cyclases and examined for activation by the RAS2 protein. We found that the fusion containing both the NH2-terminal 1600 residues and the COOH-terminal 66 residues of the Sa. cerevisiae cyclase rendered the catalytic domain of the Sc. pombe cyclase, which otherwise did not respond to RAS proteins, activatable by the RAS2 protein. Thus the leucine-rich repeats and the COOH terminus of the Sa. cerevisiae adenylate cyclase appear to be required for interaction with RAS proteins. Images PMID:2247439

  16. Enzyme Informatics

    PubMed Central

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  17. Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin.

    PubMed Central

    Gordon, V M; Leppla, S H; Hewlett, E L

    1988-01-01

    Bordetella pertussis and Bacillus anthracis produce extracytoplasmic adenylate cyclase toxins (AC toxins) with shared features including activation by calmodulin and the ability to enter target cells and catalyze intracellular cyclic AMP (cAMP) production from host ATP. The two AC toxins were evaluated for sensitivities to a series of inhibitors of known uptake mechanisms. Cytochalasin D, an inhibitor of microfilament function, abrogated the cAMP response to B. anthracis AC toxin (93%) but not the cAMP response elicited by B. pertussis AC toxin. B. anthracis-mediated intoxication of CHO cells was completely inhibited by ammonium chloride (30 mM) and chloroquine (0.1 mM), whereas the cAMP accumulation produced by B. pertussis AC toxin remained unchanged. The block of target cell intoxication by cytochalasin D could be bypassed when cells were first treated with anthrax AC toxin and then exposed to an acidic medium. These data indicate that despite enzymatic similarities, these two AC toxins intoxicate target cells by different mechanisms, with anthrax AC toxin entering by means of receptor-mediated endocytosis into acidic compartments and B. pertussis AC toxin using a separate, and as yet undefined, mechanism. PMID:2895741

  18. Pituitary adenylate cyclase-activating polypeptide (PACAP) has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models

    PubMed Central

    Kiss, Tibor; Jungling, Adel

    2017-01-01

    ABSTRACT Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP. PMID:28067625

  19. Pituitary adenylate cyclase-activating polypeptide (PACAP) has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models.

    PubMed

    Maasz, Gabor; Zrinyi, Zita; Reglodi, Dora; Petrovics, Dora; Rivnyak, Adam; Kiss, Tibor; Jungling, Adel; Tamas, Andrea; Pirger, Zsolt

    2017-02-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP.

  20. Subnanometre enzyme mechanics probed by single-molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Pelz, Benjamin; Žoldák, Gabriel; Zeller, Fabian; Zacharias, Martin; Rief, Matthias

    2016-02-01

    Enzymes are molecular machines that bind substrates specifically, provide an adequate chemical environment for catalysis and exchange products rapidly, to ensure fast turnover rates. Direct information about the energetics that drive conformational changes is difficult to obtain. We used subnanometre single-molecule force spectroscopy to study the energetic drive of substrate-dependent lid closing in the enzyme adenylate kinase. Here we show that in the presence of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A), closing and opening of both lids is cooperative and tightly coupled to inhibitor binding. Surprisingly, binding of the substrates ADP and ATP exhibits a much smaller energetic drive towards the fully closed state. Instead, we observe a new dominant energetic minimum with both lids half closed. Our results, combining experiment and molecular dynamics simulations, give detailed mechanical insights into how an enzyme can cope with the seemingly contradictory requirements of rapid substrate exchange and tight closing, to ensure efficient catalysis.

  1. Subnanometre enzyme mechanics probed by single-molecule force spectroscopy

    PubMed Central

    Pelz, Benjamin; Žoldák, Gabriel; Zeller, Fabian; Zacharias, Martin; Rief, Matthias

    2016-01-01

    Enzymes are molecular machines that bind substrates specifically, provide an adequate chemical environment for catalysis and exchange products rapidly, to ensure fast turnover rates. Direct information about the energetics that drive conformational changes is difficult to obtain. We used subnanometre single-molecule force spectroscopy to study the energetic drive of substrate-dependent lid closing in the enzyme adenylate kinase. Here we show that in the presence of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A), closing and opening of both lids is cooperative and tightly coupled to inhibitor binding. Surprisingly, binding of the substrates ADP and ATP exhibits a much smaller energetic drive towards the fully closed state. Instead, we observe a new dominant energetic minimum with both lids half closed. Our results, combining experiment and molecular dynamics simulations, give detailed mechanical insights into how an enzyme can cope with the seemingly contradictory requirements of rapid substrate exchange and tight closing, to ensure efficient catalysis. PMID:26906294

  2. Marine enzymes.

    PubMed

    Debashish, Ghosh; Malay, Saha; Barindra, Sana; Joydeep, Mukherjee

    2005-01-01

    Marine enzyme biotechnology can offer novel biocatalysts with properties like high salt tolerance, hyperthermostability, barophilicity, cold adaptivity, and ease in large-scale cultivation. This review deals with the research and development work done on the occurrence, molecular biology, and bioprocessing of marine enzymes during the last decade. Exotic locations have been accessed for the search of novel enzymes. Scientists have isolated proteases and carbohydrases from deep sea hydrothermal vents. Cold active metabolic enzymes from psychrophilic marine microorganisms have received considerable research attention. Marine symbiont microorganisms growing in association with animals and plants were shown to produce enzymes of commercial interest. Microorganisms isolated from sediment and seawater have been the most widely studied, proteases, carbohydrases, and peroxidases being noteworthy. Enzymes from marine animals and plants were primarily studied for their metabolic roles, though proteases and peroxidases have found industrial applications. Novel techniques in molecular biology applied to assess the diversity of chitinases, nitrate, nitrite, ammonia-metabolizing, and pollutant-degrading enzymes are discussed. Genes encoding chitinases, proteases, and carbohydrases from microbial and animal sources have been cloned and characterized. Research on the bioprocessing of marine-derived enzymes, however, has been scanty, focusing mainly on the application of solid-state fermentation to the production of enzymes from microbial sources.

  3. Genetical control and linkage relationships of isozyme markers in sugar beet (B. vulgaris L.) : 1. Isocitrate dehydrogenase, adenylate kinase, phosphoglucomutase, glucose phosphate isomerase and cathodal peroxidase.

    PubMed

    Smed, E; Van Geyt, J P; Oleo, M

    1989-07-01

    Five isozyme systems were genetically investigated. The different separation techniques, the developmental expression and the use as marker system in sugar beet genetics and breeding is discussed. Isocitrate dehydrogenase was controlled by two genes. The gene products form inter- as well as intralocus dimers, even with the gene products of the Icd gene in B. procumbens and B. patellaris. Adenylate kinase was controlled by one gene. Three different allelic forms were detected, which were active as monomeric proteins. Glucose phosphate isomerase showed two zones of activity. One zone was polymorphic. Three allelic variants, active as dimers, were found. Phosphoglucomutase also showed two major zones of activity. One zone was polymorphic and coded for monomeric enzymes. Two allelic forms were found in the accessions studied. The cathodal peroxidase system was controlled by two independent genes, of which only one was polymorphic. The gene products are active as monomers. Linkage was found between red hypocotyl color (R) and Icd 2. Pgm 1, Gpi 2, Ak 1 and the Icd 2-R linkage group segregated independently.

  4. Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis

    PubMed Central

    Kladova, A. V.; Gavel, O. Yu.; Mukhopaadhyay, A.; Boer, D. R.; Teixeira, S.; Shnyrov, V. L.; Moura, I.; Moura, J. J. G.; Romão, M. J.; Trincão, J.; Bursakov, S. A.

    2009-01-01

    Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfo­vibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 Å resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 Å resolution, respectively. Zn2+–AK and Fe2+–AK crystallized in space group I222 with similar unit-cell parameters, whereas Co2+–AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn2+–AK and Fe2+–AK forms and a dimer was present for the Co2+–AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes. PMID:19724135

  5. Interictal plasma pituitary adenylate cyclase-activating polypeptide levels are decreased in migraineurs but remain unchanged in patients with tension-type headache.

    PubMed

    Han, Xun; Dong, Zhao; Hou, Lei; Wan, Dongjun; Chen, Min; Tang, Wenjing; Yu, Shengyuan

    2015-10-23

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is associated with migraine phase; however, whether PACAP levels could be used to distinguish between migraine and tension-type headache (TTH) remains unknown. We compared interictal plasma PACAP levels among healthy controls, migraineurs, and patients with TTH. Interictal plasma levels of PACAP were measured in 133 migraineurs, 106 patients with TTH, and 50 controls using enzyme-linked immunoassays. We further evaluated the relationships between interictal PACAP plasma concentrations and clinical parameters, such as headache severity, attack frequency, and duration. We found that migraineurs had significantly lower interictal plasma PACAP levels than patients with TTH and healthy controls. However, there were no significant differences between patients with TTH and healthy controls. Plasma PACAP levels were significantly lower in patients with episodic migraine (EM) than in patients with episodic tension-type headache (ETTH) and in patients with chronic migraine (CM) than in patients with chronic tension-type headache (CTTH). Interictal PACAP levels were negatively correlated with duration in the CM group. The results of this study demonstrated differences in interictal PACAP levels in migraine and TTH, suggesting that PACAP is involved in the pathogenesis of migraine rather than TTH. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis.

    PubMed

    Kladova, A V; Gavel, O Yu; Mukhopaadhyay, A; Boer, D R; Teixeira, S; Shnyrov, V L; Moura, I; Moura, J J G; Romão, M J; Trincão, J; Bursakov, S A

    2009-09-01

    Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfovibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 A resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 A resolution, respectively. Zn(2+)-AK and Fe(2+)-AK crystallized in space group I222 with similar unit-cell parameters, whereas Co(2+)-AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn(2+)-AK and Fe(2+)-AK forms and a dimer was present for the Co(2+)-AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes.

  7. Cyclic adenosine 3',5'-monophosphate levels and activities of adenylate cyclase and cyclic adenosine 3',5'-monophosphate phosphodiesterase in Pseudomonas and Bacteroides.

    PubMed Central

    Siegel, L S; Hylemon, P B; Phibbs, P V

    1977-01-01

    A modified Gilman assay was used to determine the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) in rapidly filtered cells and in the culture filtrates of Pseudomonas aeruginosa, Escherichia coli K-12, and Bacteroides fragilis. In P. aeruginosa cultures, levels of cAMP in the filtrate increased with the culture absorbance (3.5 to 19.8 X 10(-9) M) but did not vary significantly with the carbon source used to support growth. Intracellular concentrations (0.8 to 3.2 X 10(-5) M) were substantially higher and did not vary appreciably during growth or with carbon source. Sodium cAMP (5 mM) failed to reverse the catabolite repression of inducible glucose-6-phosphate dehydrogenase (EC 1.1.1.49) synthesis caused by the addition of 10 mM succinate. Exogenous cAMP also had no discernible effect on the catabolite repression control of inducible mannitol dehydrogenase (EC 1.1.1.67). P. aeruginosa was found to contain both soluble cAMP phosphodiesterase (EC 3.1.4.17) and membrane-associated adenylate cyclase (EC 4.6.1.1) activity, and these were compared to the activities detected in crude extracts of E. coli. B. fragilis crude cell extracts contain neither of these enzyme activities, and little or no cAMP was detected in cells or culture filtrates of this anaerobic bacterium. PMID:187575

  8. Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

    PubMed Central

    Zhang, Keya; Nelson, Kathryn M.; Bhuripanyo, Karan; Grimes, Kimberly D.; Zhao, Bo; Aldrich, Courtney C.; Yin, Jun

    2013-01-01

    SUMMARY The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the “nonribosomal code” of A-domains. PMID:23352143

  9. Alterations in adenylate ratios in plant cells after accelerated ion irradiation

    NASA Astrophysics Data System (ADS)

    Vasilenko, A.; Sidorenko, P. G.

    Levels of adenylate metabolism have been studied in cells of Nicotiana tabacum growing in vitro, and in root apex extracts of Pisum sativum irradiated at the 95-in. isochronous cyclotron U-240, Institute for Nuclear Research, Ukrainian National Academy of Sciences, Kyiv. Particle beams of accelerated helium ions with energy 9.34 keV/mum were used. Replacement and rapid freezing of the irradiated plant samples in liquid nitrogen were carried out with a manipulator and a remote control system. After doses of 5, 20, 50, and 100 Gy of gamma-irradiation, as well as 50 and 100 Gy ^4He irradiation, the cellular ATP/ADP ratio increased during early stages of the response. This effect was absent at higher doses and after exposure to sparsely-ionizing radiation, when a rapid decline in the cellular ATP concentration and the ATP/ADP ratio occurred.

  10. Role of the adenylate energy charge in the response of Chinese hamster ovary cells to radiation

    SciTech Connect

    Bump, E.A.; Calderwood, S.K.; Sawyer, J.M.; Brown, J.M.

    1984-08-01

    Steady-state modification of the adenylate energy charge in aerobic Chinese hamster ovary (CHO) cells was achieved with a combination of rotenone and 2-deoxy glucose (2dG). The radiation response of these cells was not significantly affected by this treatment when cells were irradiated either one or three hours after addition of the drugs, and held in the presence of the inhibitors for one hour after irradiation. The ability of cells to repair radiation-induced single-strand breaks was studied by the alkaline elution method. Energy depleted cells repaired single-strand breaks at a slightly slower rate than the controls. However, thymidine incorporation was also inhibited, suggesting that repair may still have preceded events leading to the fixation of that damage (e.g., DNA replication).

  11. Pertussis toxin and adenylate cyclase toxin: key virulence factors of Bordetella pertussis and cell biology tools

    PubMed Central

    Carbonetti, Nicholas H

    2010-01-01

    Pertussis toxin and adenylate cyclase toxin are two important virulence factors of Bordetella pertussis, the bacterial cause of the respiratory disease pertussis or whooping cough. In addition to studies on the structure, function and role in pathogenesis of these two toxins, they are both used as cell biology tools for a variety of applications owing to their ability to enter mammalian cells, perform enzymatic activities and modify cell signaling events. In this article, recent data from the research literature that enhance our understanding of the nature of these two toxins, their role in the pathogenesis of B. pertussis infection and disease, particularly in modulating host immune responses, and their use as tools for other areas of research will be outlined. PMID:20210554

  12. Active-site modifications of adenylation domains lead to hydrolysis of upstream nonribosomal peptidyl thioester intermediates.

    PubMed

    Uguru, Gabriel C; Milne, Claire; Borg, Matthew; Flett, Fiona; Smith, Colin P; Micklefield, Jason

    2004-04-28

    Site-directed mutagenesis of nonribosomal peptide synthetase (NRPS) adenylation (A) domains was investigated as a means to engineer new calcium-dependent antibiotics (CDA) in Streptomyces coelicolor. Single- and double-point mutants of the CDA NRPS module 7, A-domain were generated, which were predicted to alter the specificity of this domain from Asp to Asn. The double-point mutant produced a new peptide CDA2a-7N containing Asn at position 7 as expected. However, in both the single- and the double-point mutants, significant hydrolysis of the CDA-6mer intermediate was evident. One explanation for this is that the mutant module 7 A-domain activates Asn instead of Asp; however, the Asn-thioester intermediate is only weakly recognized by the upstream C-domain acceptor site (a), allowing a water molecule to intercept the hexapeptidyl intermediate in the donor site (d).

  13. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles.

    PubMed

    Donato, Gina M; Goldsmith, Cynthia S; Paddock, Christopher D; Eby, Joshua C; Gray, Mary C; Hewlett, Erik L

    2012-02-17

    Bordetella pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B. pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B. pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin's receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV-ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV-ACT can deliver ACT by processes distinct from those of ACT alone. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

  14. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles

    PubMed Central

    Donato, Gina M.; Goldsmith, Cynthia S.; Paddock, Christopher D.; Eby, Joshua C.; Gray, Mary C.; Hewlett, Erik L.

    2012-01-01

    B.pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B.pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B.pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin’s receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV-ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV-ACT can deliver ACT by processes distinct from those of ACT alone. PMID:22289177

  15. The influence of various cations on the catalytic properties of clays. [polymerization of alanine adenylate

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1978-01-01

    The polymerization of alanine adenylate in the presence of the sodium form of various clays was studied, and hectorite was found to cause more polymerization than nontronite and montmorillonite (in that order) although the differences were not great. The effect on polymerization of presaturating montmorillonite with different cations was determined. Hectorite, with increased basicity of the interspatial planes, allows polymerization of lysine, which montmorillonite does not. The general trend is that, for the same amino acid, higher degrees of polymerization are obtained when the cation in the octahedral lattice of the clay is divalent rather than trivalent. With the exchangeable cations the order is reversed, for a reason that is explained. The main role of clays in the polymerization mechanism of amino acids is concentration and neutralization of charges.

  16. Adenylate cyclase 5 is required for melanophore and male pattern development in the guppy (Poecilia reticulata).

    PubMed

    Kottler, Verena A; Künstner, Axel; Koch, Iris; Flötenmeyer, Matthias; Langenecker, Tobias; Hoffmann, Margarete; Sharma, Eshita; Weigel, Detlef; Dreyer, Christine

    2015-09-01

    Guppies (Poecilia reticulata) are colorful fish that have attracted the attention of pigmentation researchers for almost a century. Here, we report that the blond phenotype of the guppy is caused by a spontaneous mutation in the guppy ortholog of adenylate cyclase 5 (adcy5). Using double digest restriction site-associated DNA sequencing (ddRADseq) and quantitative trait locus (QTL) mapping, we linked the blond phenotype to a candidate region of 118 kb, in which we subsequently identified a 2-bp deletion in adcy5 that alters splicing and leads to a premature stop codon. We show that adcy5, which affects life span and melanoma growth in mouse, is required for melanophore development and formation of male orange pigmentation traits in the guppy. We find that some components of the male orange pattern are particularly sensitive to loss of Adcy5 function. Our work thus reveals a function for Adcy5 in patterning of fish color ornaments.

  17. Cannabinoid inhibition of adenylate cyclase: relative activity of constituents and metabolites of marihuana.

    PubMed

    Howlett, A C

    1987-05-01

    delta 9Tetrahydrocannabinol (THC) has been shown to inhibit the activity of adenylate cyclase in the N18TG2 clone of murine neuroblastoma cells. The concentration of delta 9THC exhibiting half-maximal inhibition was 500 nM. delta 8Tetrahydrocannabinol was less active, and cannabinol was only partially active. Cannabidiol, cannabigerol, cannabichromene, olivetol and compounds having a reduced length of the C3 alkyl side chain were inactive. The metabolites of delta 8THC and delta 9THC hydroxylated at the C11 position were more potent than the parent drugs. However, hydroxylation at the C8 position of the terpenoid ring resulted in loss of activity. Compounds hydroxylated along the C3 alkyl side chain were equally efficacious but less potent than delta 9THC. These findings are compared to the pharmacology of cannabinoids reported for psychological effects in humans and behavioral effects in a variety of animal models.

  18. Tachyphylaxis to PACAP-27 after inhibition of NO synthesis: a loss of adenylate cyclase activation

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The vasodilator effects of pituitary adenylate cyclase activating polypeptide (PACAP-27) are subject to tachyphylaxis in rats treated with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). This study examined whether this tachyphylaxis is due to the loss of vasodilator potency of cAMP generated by activation of the G(s) protein-coupled PACAP receptors. Five successive treatments with PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats that were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME (50 micromol/kg iv)-treated rats produced vasodilator responses of similar magnitude to those in saline-treated rats, whereas four subsequent injections produced progressively and markedly smaller responses. The hemodynamic effects of the membrane-permeable cAMP analog 8-(4-chlorophenylthiol)-cAMP (8-CPT-cAMP; 5-15 micromol/kg iv) were similar in L-NAME-treated rats and in L-NAME-treated rats that had received the five injections of PACAP-27. In addition, five injections of 8-CPT-cAMP (10 micromol/kg iv) produced pronounced vasodilator responses in saline- and L-NAME-treated rats that were not subject to the development of tachyphylaxis. These results suggest that a loss of biological potency of cAMP is not responsible for tachyphylaxis to PACAP-27 in L-NAME-treated rats. This tachyphylaxis may be due to the inability of the G(s) protein-coupled PACAP receptor to activate adenylate cyclase.

  19. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    NASA Astrophysics Data System (ADS)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-12-01

    We studied the spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay. This led to the unexpected finding that the degrees of polymerization (DP's) of the oligo- and poly-peptides obtained depended on the amounts of polypeptides that were preadsorbed. Plotting average molecular weights obtained against c-spacings of the clay platelet aggregates which widened as a result of polypeptide addition and adsorption before the polymerization, does not permit an obvious explanation of these observations. The best correlation assigns a role to the fractional occupation of the individual intercalation layers of the polypeptides, as the adsorption increases towards a first complete mono-interlayer, then to an incipient and eventually to a complete double layer on to a third interlayer, after which the clay stacking breaks up. Spacings which correspond to an intermediate occupation of any of the three successive interlayers favor amino acids self-addition to polymers. The opposite is true for nearly empty or filled intercalation layers. We hypothesize and describe, how a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances. Moderately filled intercalation spaces may also act as sinks for the newly formed oligomers and facilitate the freeing of reaction sites for the occupation by fresh reagent. The c-spacings required for these mechanisms are the result of the intercalation of the preadsorbed polymer, but similar conditions prevail when polymers are adsorbed as they are generated during polymerization.

  20. Meglumine cyclic adenylate improves neurological function following acute spinal cord injury in rats.

    PubMed

    Liao, Jingwu; Xie, Jingming; Lin, Daqiang; Lu, Ning; Guo, Limin; Li, Weiqiang; Pu, Bo; Yang, Yang; Yang, Zhenlong; Zhang, Ying; Song, Yueming

    2014-09-01

    Elevation of intracellular cyclic adenosine monophosphate (cAMP) levels facilitates recovery following spinal injury by suppressing secondary pathology and promoting axonal regeneration. However, this treatment strategy is limited by lack of effective and tolerable clinical agents. The present study examined the effects of meglumine cyclic adenylate (MCA) on neurological recovery, cAMP concentration, adenylate cyclase 3 (AC3) activity and phosphodiesterase 4D (PDE4D) activity during early stage acute spinal cord injury (SCI) in rats. A total of 48 Sprague‑Dawley rats were randomly assigned to groups A, B or C, each consisting of 16 animals. SCI was induced by Allen's method using a 7 g x 3 cm extradural weight‑drop impact on spinal cord segment T11. A total of 30 min following SCI, group A received a single 30 mg/kg‑bw i.p. dose of methylprednisolone, group B received 2 mg/kg‑bw i.p. MCA daily for seven days and group C were administered an equal volume of normal saline. Seven days following SCI, the spinal cord samples from eight rats per group were obtained to measure the cAMP concentration, and the activities of AC3 and PDE4D. The remaining eight rats per group were used for behavioral assessments using the inclined plane stability test and Gale scale for up to six weeks post‑SCI. The drug‑treated groups A and B had higher cAMP concentrations and AC3 activities but lower PDE4D activities at the lesion sites, as well as superior behavioral scores post‑SCI compared with the vehicle‑treated group C (P<0.05). Furthermore, cAMP was higher in group B than in group A (P<0.05). It was concluded that MCA may serve as an effective SCI treatment by activating AC3 and suppressing PDE4D.

  1. Characterization of beta-adrenergic receptors and adenylate cyclase activity in rat brown fat

    SciTech Connect

    Baresi, L.A.; Morley, J.E.; Scarpace, P.J.

    1986-03-01

    Catecholamines stimulate thermogenesis in rat brown fat through a mechanism which involves binding to the beta-adrenergic receptor (BAR), stimulation of adenylate cyclase (AC) and culminating with uncoupling of mitochondrial respiration from ATP synthesis. The authors characterized BAR, AC and cytochrome (cyt) c oxidase in CDF (F-344) interscapular brown fat. Scatchard analysis of (/sup 125/)Iodopindolol binding yields a straight line consistent with a single class of antagonist binding sites with 41.8 +/- 12.0 fmol BAR/mg protein and a K/sub d/ of 118 +/- 15 pM. Binding was both specific and stereospecific. Competition with 1-propranolol (K/sub d/ = 6.7 nM) was 15 times more potent than d-propranolol (K/sub d/ = 103 nM). Competition with isoproterenol (K/sub d/ = 79 nM) was 10 times more potent than epinephrine (K/sub d/ = 820 nM) which was 35 times more potent than norepinephrine (K/sub d/ = 2.9 x 10/sup -5/ M) suggesting predominate beta/sub 2/-type BAR. Cyt c oxidase activity was assessed in brown fat mitochrondrial preparations. The ratio of BAR to cyt c activity was 959 +/- 275 nmol BAR/mol cyc c/min. Isoproterenol (0.1 mM) stimulated AC activity was 24 times GTP (0.1 mM) stimulated AC (98.5 vs 40.7 pmol cAMP/min/mg). NaF-stimulated AC was nine times basal activity (90.5 vs 11.3 pmol cAMP/min/mg). These data demonstrate the presence of a beta-/sub 2/-type BAR coupled to adenylate cyclase in rat brown fat.

  2. New crystal structures of adenylate kinase from Streptococcus pneumoniae D39 in two conformations.

    PubMed

    Thach, Trung Thanh; Lee, Sangho

    2014-11-01

    Adenylate kinases (AdKs; EC 2.7.3.4) play a critical role in intercellular homeostasis by the interconversion of ATP and AMP to two ADP molecules. Crystal structures of adenylate kinase from Streptococcus pneumoniae D39 (SpAdK) have recently been determined using ligand-free and inhibitor-bound crystals belonging to space groups P21 and P1, respectively. Here, new crystal structures of SpAdK in ligand-free and inhibitor-bound states determined at 1.96 and 1.65 Å resolution, respectively, are reported. The new ligand-free crystal belonged to space group C2, with unit-cell parameters a=73.5, b=54.3, c=62.7 Å, β=118.8°. The new ligand-free structure revealed an open conformation that differed from the previously determined conformation, with an r.m.s.d on Cα atoms of 1.4 Å. The new crystal of the complex with the two-substrate-mimicking inhibitor P1,P5-bis(adenosine-5'-)pentaphosphate (Ap5A) belonged to space group P1, with unit-cell parameters a=53.9, b=62.3, c=63.0 Å, α=101.9, β=112.6, γ=89.9°. Despite belonging to the same space group as the previously reported crystal, the new Ap5A-bound crystal contains four molecules in the asymmetric unit, compared with two in the previous crystal, and shows slightly different lattice contacts. These results demonstrate that SpAdK can crystallize promiscuously in different forms and that the open structure is flexible in conformation.

  3. Synthetic genes for human muscle-type adenylate kinase in Escherichia coli.

    PubMed

    Kim, H J; Nishikawa, S; Tanaka, T; Uesugi, S; Takenaka, H; Hamada, M; Kuby, S A

    1989-01-01

    An artificial gene coding for the human muscle-type cytosolic adenylate kinase (hAK1) was chemically synthesized and directly expressed in Escherichia coli under the control of trp promoter. The DNA duplex of 596 bp was designed and constructed from 40 oligonucleotide fragments of typically 30 nucleotides in length. Twelve unique restriction sites were fairly evenly spaced in the synthetic gene to facilitate site-specific mutagenesis at any part of this recombinant protein. The genes for mutant hAK1 (Tyr 95----Phe 95, Y95F hAK1; Arg 97----Ala 97, R97A hAK1) were constructed by cassette mutagenesis and utilized restriction sites incorporated in the hAK1 gene. The recombinant hAK1 was purified to homogeneity by a two-step chromatographic procedure with a good yield, and showed the same adenylate kinase activity as that of authentic hAK1. Preliminary kinetic studies show that the enzymatic activity (Vmax app,cor/Et) of Y95F hAK1 was slightly greater than that of recombinant hAK1, whereas R97A hAK1 still possessed approximately 4% of recombinant hAK1 activity. These results suggest that the Arg-97 residue is important but not essential for catalytic activity, and that Tyr-95 can be replaced by phenylalanine without substantial effects on the enzymatic activity. Moreover, preliminary estimates of the apparent kinetic parameters suggest that these residues are not required for MgATP binding, and therefore they do not appear to be part of the MgATP binding site.

  4. Understanding Enzymes.

    ERIC Educational Resources Information Center

    Sinnott, M. L.

    1979-01-01

    Describes the way enzymes operate through reaction energetics, and explains that most of the catalytic power of enzymes lies in the strong noncovalent forces responsible for initial binding of substrate, which are only manifested at the transition state of the reaction. (Author/GA)

  5. Enzymes, Industrial

    USDA-ARS?s Scientific Manuscript database

    Enzymes serve key roles in numerous biotechnology processes and products that are commonly encountered in the forms of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes can display regio- and stereo-specificity, p...

  6. A simple enzymic method for the synthesis of adenosine 5'-[alpha-32P]triphosphate on a preparative scale.

    PubMed

    Martin, B R; Voorheis, H P

    1977-03-01

    A simple, rapid and inexpensive method is described for the enzymic synthesis of [alpha-32P]ATP from [32P]Pi on a preparative scale with an overall yield of 53%. The final product contained all of the detectable radioactivity (less than 99.9%) in the alpha position and has been shown to behave identically with commerically availabe [alpha-32P]ATP during the synthesis of 3':5'-cyclic AMP in the reaction catalysed by adenylate cyclase.

  7. Thermostable adenylate kinase technology: a new process indicator and its use as a validation tool for the reprocessing of surgical instruments.

    PubMed

    Hesp, J R; Poolman, T M; Budge, C; Batten, L; Alexander, F; McLuckie, G; O'Brien, S; Wells, P; Raven, N D H; Sutton, J M

    2010-02-01

    Adenylate kinase (tAK), a thermostable enzyme, was assessed as a possible means of providing a quantitative measure of cleaning efficacy suitable for validating the performance of an automated washer disinfector (AWD) during routine use. Two indicator formulations were developed using either a commercially available washer disinfector soil or a protein-based soil. Each indicator consisted of 100 microg (in test soil) of tAK dried on to a steel or plastic surface. These indicators were placed in each basket of a washer disinfector and processed alongside soiled surgical instruments during a standard day's operation. After processing, remaining tAK activity was detected using a rapid enzyme assay (2 min detection time) in a handheld hygiene monitor. The amount of tAK remaining on each indictor after a full AWD cycle was found to range from 0.1 to 0.4 ng, which represented a mean log(10) removal of 5.8+/-0.3. There was no statistical difference in the residual tAK activity between individual runs or the position of the indicator in the machine. The tAK indicator was also used to analyse the protein removal within each component of the wash cycle. These results demonstrated that all phases of the wash process contributed to the removal of the protein load, with the main wash alone being responsible for 3.6-4.0 log(10) reductions in protein activity. We propose that a quantitative cleaning index using such rapid readout indicator devices would provide a valuable addition to the methodologies for validating cleaning processes.

  8. A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

    PubMed Central

    2012-01-01

    Background Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available. Results We demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum) using the T4 RNA ligase 1 adenylated adapter. Conclusion We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples. PMID:22995534

  9. Cu-free cycloaddition for identifying catalytic active adenylation domains of nonribosomal peptide synthetases by phage display.

    PubMed

    Zou, Yekui; Yin, Jun

    2008-10-15

    To engineer the substrate specificities of nonribosomal peptide synthetases (NRPS), we developed a method to display NRPS modules on M13 phages and select catalytically active adenylation (A) domains that would load azide functionalized substrate analogs to the neighboring peptidyl carrier protein (PCP) domains. Biotin conjugated difluorinated cyclooctyne was used for copper free cycloaddition with an azide substituted substrate attached to PCP. Biotin-labeled phages were selected by binding to streptavidin.

  10. Adenylate pool and energy charge in human lymphocytes and granulocytes irradiated at 632 nm (HeNe laser)

    NASA Astrophysics Data System (ADS)

    Bolognani, Lorenzo; Venturelli, T.; Volpi, N.; Zirilli, O.

    1995-05-01

    Aim of this report was to investigate the adenylate pool and the energy charge in human white blood cells exposed to increasing time (15, 30 and 60 min) of HeNe laser treatment. EDTA treated human blood diluted 1:1 with 0.88% KCl was added (1:5) with NaCl-dextran solution to allow sedimentation of red blood cells. 6 ml of the white cells floating in the supernatant were layered on 3 ml of Lymphoprep in plastic tubes and each tube was centrifuged (from 50 to 5000 X g for 5 min). Granulocytes were concentrated in the lower phase, whilst lymphocytes were in the intermediated phase. After further purification cytological homogeneity was tested by a cell counter. Granulocytes and lymphocytes were irradiated at +22°C with HeNe (Space, Valfivre equipment). On these population ATP was tested by luminometric procedure, the adenylate pool was separated by HPLC (Jasco) on neutralyzed perchloric extracts. ATP concentration increased in lymphocytes (+63.9%, p < 0.01) and in granulocytes (+25.0%, p < 0.05) after 60 min irradiation. The adenylate pool (tested by HPLC) does not change significatively in lymphocytes or granulocytes after 30 min irradiation, whilst in 60 min irradiated lymphocytes and granulocytes a significative increment was observed in nucleotide concentration. No changes were observed in energy charge according to Atkinson.

  11. Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    SciTech Connect

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1987-05-01

    The purpose of the present investigation was to determine if dietary lipids can induce changes in the adenylate cyclase system in rat heart. Three groups of male young Sprague-Dawley rats were fed for 6 weeks diets containing 10% corn oil (I), 8% coconut oil + 2% corn oil (II) or 10% menhaden oil (III). Adenylate cyclase activity (basal, fluoride-, isoproterenol-, and forskolin-stimulated) was higher in heart homogenates of rats in group III than in the other two groups. Concentration of the (/sup 3/H)-forskolin binding sites in the cardiac membranes were significantly higher in rats fed menhaden oil. The values (pmol/mg protein) were 4.8 +/- 0.2 (I), 4.5 +/- 0.7 (II) and 8.4 +/- 0.5 (III). There was no significant difference in the affinity of the forskolin binding sites among the 3 dietary groups. When measured at different concentrations of forskolin, the adenylate cyclase activity in cardiac membranes of rats fed menhaden oil was higher than in the other 2 groups. Concentrations of the (/sup 3/H)DHA binding sites were slightly higher but their affinity was lower in cardiac membranes of rats fed menhaden oil. The results suggest that diets containing fish oil increase the concentration of the forskolin binding sites and may also affect the characteristics of the ..beta..-adrenergic receptor in rat heart.

  12. Mechanism of activation of light-activated phosphodiesterase and evidence for homology with hormone-activated adenylate cyclase

    SciTech Connect

    Bitensky, M.W.; Yamazaki, A.; Wheeler, M.A.; George, J.S.; Rasenick, M.M.

    1983-01-01

    Light-activated cGMP phosphodiesterase (PDE) is one of the effector proteins in the rod outer segments in vertebrate retina. The hydrolysis of cGMP in rod occurs with a speed and light sensitivity which suggests a role for this hydrolysis in visual transduction. In fact, there is electrophysiological data which supports the possibility that cGMP could regulate rod membrane voltage. PDE shows very rapid activation in the presence of photons and GTP. We have called attention to the intriguing analogy between light activated rod phosphodiesterase and hormone activated adenylate cyclase. A number of studies have implicated the binding of GTP to a GTP binding protein as a factor in the hormone dependent activation of adenylate cyclase. Moreover, Cassel and Selinger have shown that hydrolysis of GTP is a component in the inactivation of the hormone dependent adenylate cyclase. We review here recent additional data which provide specific molecular details of the mechanism of light activation of rod PDE as well as demonstrate the exchange of components between light activated PDE and hormone activated cyclase.

  13. Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB. 20

    SciTech Connect

    Conner, D.A.

    1988-01-01

    Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT{sub 1C} receptor, which modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing ({sup 3}H)serotonin, ({sup 3}H)lysergic acid diethylamide or ({sup 3}H)dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor.

  14. High throughput synthetic lethality screen reveals a tumorigenic role of adenylate cyclase in fumarate hydratase-deficient cancer cells

    PubMed Central

    2014-01-01

    Background Synthetic lethality is an appealing technique for selectively targeting cancer cells which have acquired molecular changes that distinguish them from normal cells. High-throughput RNAi-based screens have been successfully used to identify synthetic lethal pathways with well-characterized tumor suppressors and oncogenes. The recent identification of metabolic tumor suppressors suggests that the concept of synthetic lethality can be applied to selectively target cancer metabolism as well. Results Here, we perform a high-throughput RNAi screen to identify synthetic lethal genes with fumarate hydratase (FH), a metabolic tumor suppressor whose loss-of-function has been associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Our unbiased screen identified synthetic lethality between FH and several genes in heme metabolism, in accordance with recent findings. Furthermore, we identified an enrichment of synthetic lethality with adenylate cyclases. The effects were validated in an embryonic kidney cell line (HEK293T) and in HLRCC-patient derived cells (UOK262) via both genetic and pharmacological inhibition. The reliance on adenylate cyclases in FH-deficient cells is consistent with increased cyclic-AMP levels, which may act to regulate cellular energy metabolism. Conclusions The identified synthetic lethality of FH with adenylate cyclases suggests a new potential target for treating HLRCC patients. PMID:24568598

  15. High throughput synthetic lethality screen reveals a tumorigenic role of adenylate cyclase in fumarate hydratase-deficient cancer cells.

    PubMed

    Boettcher, Michael; Lawson, Andrew; Ladenburger, Viola; Fredebohm, Johannes; Wolf, Jonas; Hoheisel, Jörg D; Frezza, Christian; Shlomi, Tomer

    2014-02-25

    Synthetic lethality is an appealing technique for selectively targeting cancer cells which have acquired molecular changes that distinguish them from normal cells. High-throughput RNAi-based screens have been successfully used to identify synthetic lethal pathways with well-characterized tumor suppressors and oncogenes. The recent identification of metabolic tumor suppressors suggests that the concept of synthetic lethality can be applied to selectively target cancer metabolism as well. Here, we perform a high-throughput RNAi screen to identify synthetic lethal genes with fumarate hydratase (FH), a metabolic tumor suppressor whose loss-of-function has been associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Our unbiased screen identified synthetic lethality between FH and several genes in heme metabolism, in accordance with recent findings. Furthermore, we identified an enrichment of synthetic lethality with adenylate cyclases. The effects were validated in an embryonic kidney cell line (HEK293T) and in HLRCC-patient derived cells (UOK262) via both genetic and pharmacological inhibition. The reliance on adenylate cyclases in FH-deficient cells is consistent with increased cyclic-AMP levels, which may act to regulate cellular energy metabolism. The identified synthetic lethality of FH with adenylate cyclases suggests a new potential target for treating HLRCC patients.

  16. Structure of the RNA 30-Phosphate Cyclase-Adenylate Intermediate Illuminates Nucleotide Specificity and Covalent Nucleotidyl Transfer

    SciTech Connect

    Tanaka, N.; Smith, P; Shuman, S

    2010-01-01

    RNA 3-phosphate cyclase (RtcA) synthesizes RNA 2,3 cyclic phosphate ends via three steps: reaction with ATP to form a covalent RtcA-AMP intermediate; transfer of adenylate to an RNA 3-phosphate to form RNA(3)pp(5)A; and attack of the vicinal O2 on the 3-phosphorus to form a 2,3 cyclic phosphate. Here we report the 1.7 {angstrom} crystal structure of the RtcA-AMP intermediate, which reveals the mechanism of nucleotidyl transfer. Adenylate is linked via a phosphoamide bond to the His309 N{var_epsilon} atom. A network of hydrogen bonds to the ribose O2 and O3 accounts for the stringent ribonucleotide preference. Adenine is sandwiched in a hydrophobic pocket between Tyr284 and Pro131 and the preference for adenine is enforced by Phe135, which packs against the purine C2 edge. Two sulfates bound near the adenylate plausibly mimic the 3-terminal and penultimate phosphates of RNA. The structure illuminates how the four {alpha}2/{beta}4 domains contribute to substrate binding and catalysis.

  17. Development of a novel photoreactive calmodulin derivative: Cross-linking of purified adenylate cyclase from bovine brain

    SciTech Connect

    Harrison, J.K.; Lawton, R.G.; Gnegy, M.E. )

    1989-07-11

    A novel photoreactive calmodulin (CaM) derivative was developed and used to label the purified CaM-sensitive adenylate cyclase from bovine cortex. {sup 125}I-CaM was conjugated with the heterobifunctional cross-linking agent p-nitrophenyl 3-diazopyruvate (DAPpNP). Spectral data indicated that diazopyruvoyl (DAP) groups were incorporated into the CaM molecule. Iodo-CaM-DAPs behaved like native CaM with respect to (1) Ca{sup 2+}-dependent enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and (2) Ca{sup 2+}-dependent stimulation of adenylate cyclase activity. {sup 125}I-CaM-DAP photochemically cross-linked to CaM-binding proteins in a manner that was both Ca{sup 2+} dependent and CaM specific. Photolysis of forskolin-agarose-purified adenylate cyclase from bovine cortex with {sup 125}I-CaM-DAP produced a single cross-linked product which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of approximately 140,000.

  18. The activity of dopamine-stimulated adenylate cyclase from rat brain striatum is modulated by temperature and the bilayer-fluidizing agent, benzyl alcohol

    PubMed Central

    Needham, Lindsey; Houslay, Miles D.

    1982-01-01

    Benzyl alcohol achieved a marked activation of the adenylate cyclase activity in a partially purified membrane preparation from rat brain striata, although inhibition resulted at high concentrations. The degree of activation observed depended on the ligand used to stimulate the enzyme, with that observed in the presence of guanosine 5′-[β,γ-imido]triphosphate (p[NH]ppG) (5.8-fold)>dopamine+p[NH]ppG (5-fold)> GTP (3-fold)>dopamine+high GTP (2.25-fold)>dopamine (+low GTP)=basal (+low GTP) (1.7-fold). The differences in the concentration-dependence of both the activation and inhibition of dopamine-stimulated and basal activities of the enzyme meant that increasing benzyl alcohol concentrations caused a net elevation in the fold-stimulation of the basal activity by dopamine. Arrhenius plots of p[NH]ppG-, GTP-, fluoride-, dopamine-plus-high GTP- and dopamine-plus-p[NH]ppG-stimulated activities all exhibited a single break occurring at around 22°C. This break point was decreased to around 13°C when 50mm-benzyl alcohol was added to the assays. In the presence of dopamine (+low GTP), Arrhenius plots exhibited two distinct breaks, one at around 21°C and the other at around 11°C. When benzyl alcohol (50mm) was added to these assays of dopamine (+low GTP)-stimulated activity, a single break at around 14°C was observed. For the basal activity the Arrhenius plot exhibited a single break at around 15°C both in the presence and in the absence of 50mm-benzyl alcohol. It is suggested that the enzyme is activated by productive collisions between independent mobile entities and that the activity of the enzyme may be regulated by changes in membrane fluidity. The breaks in the Arrhenius plots of all of the ligand-stimulated activities, but not the basal activity, are attributed to lipid-phase separations occurring in either the inner or the outer halves of the bilayer. PMID:7126197

  19. The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid.

    PubMed Central

    Berthet-Colominas, C; Seignovert, L; Härtlein, M; Grotli, M; Cusack, S; Leberman, R

    1998-01-01

    The crystal structure of Thermus thermophilus asparaginyl-tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 A resolution. This is the last of the three class IIb aminoacyl-tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl-tRNA synthetase and aspartyl-tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 A of asparaginyl-tRNA synthetase complexed with a non-hydrolysable analogue of asparaginyl-adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl-tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl-tRNA synthetase-ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl- and aspartyl-tRNA synthetases as well as that of both enzymes to the ammonia-dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor. PMID:9582288

  20. Overexpression of adenylate cyclase-associated protein 2 is a novel prognostic marker in malignant melanoma.

    PubMed

    Masugi, Yohei; Tanese, Keiji; Emoto, Katsura; Yamazaki, Ken; Effendi, Kathryn; Funakoshi, Takeru; Mori, Mariko; Sakamoto, Michiie

    2015-12-01

    Malignant melanoma is one of the lethal malignant tumors worldwide. Previously we reported that adenylate cyclase-associated protein 2 (CAP2), which is a well-conserved actin regulator, was overexpressed in hepatocellular carcinoma; however, CAP2 expression in other clinical cancers remains unclear. The aim of the current study was to clarify the clinicopathological significance of CAP2 overexpression in malignant melanoma. Immunohistochemical analyses revealed that many melanoma cells exhibited diffuse cytoplasmic expression of CAP2, whereas no normal melanocytes showed detectable immunostaining for CAP2. A high level of CAP2 expression was seen in 14 of 50 melanomas and was significantly correlated with greater tumor thickness and nodular melanoma subtypes. In addition, a high level of CAP2 expression was associated with poor overall survival in univariate and multivariate analyses. For 13 patients, samples of primary and metastatic melanoma tissue were available: four patients exhibited higher levels of CAP2 expression in metastatic tumor compared to the primary site, whereas no patient showed lower levels of CAP2 expression in metastatic melanomas. Our findings show that CAP2 overexpression is a novel prognostic marker in malignant melanoma and that CAP2 expression seems to increase stepwise during tumor progression, suggesting the involvement of CAP2 in the aggressive behavior of malignant melanoma.

  1. Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility.

    PubMed

    Yamazaki, Ken; Takamura, Masaaki; Masugi, Yohei; Mori, Taisuke; Du, Wenlin; Hibi, Taizo; Hiraoka, Nobuyoshi; Ohta, Tsutomu; Ohki, Misao; Hirohashi, Setsuo; Sakamoto, Michiie

    2009-04-01

    Pancreatic cancer has the worst prognosis among cancers due to the difficulty of early diagnosis and its aggressive behavior. To characterize the aggressiveness of pancreatic cancers on gene expression, pancreatic cancer xenografts transplanted into severe combined immunodeficient mice served as a panel for gene-expression profiling. As a result of profiling, the adenylate cyclase-associated protein 1 (CAP1) gene was shown to be overexpressed in all of the xenografts. The expression of CAP1 protein in all 73 cases of pancreatic cancer was recognized by immunohistochemical analyses. The ratio of CAP1-positive tumor cells in clinical specimens was correlated with the presence of lymph node metastasis and neural invasion, and also with the poor prognosis of patients. Immunocytochemical analyses in pancreatic cancer cells demonstrated that CAP1 colocalized to the leading edge of lamellipodia with actin. Knockdown of CAP1 by RNA interference resulted in the reduction of lamellipodium formation, motility, and invasion of pancreatic cancer cells. This is the first report demonstrating the overexpression of CAP1 in pancreatic cancers and suggesting the involvement of CAP1 in the aggressive behavior of pancreatic cancer cells.

  2. Adenylate kinase 2 (AK2) promotes cell proliferation in insect development

    PubMed Central

    2012-01-01

    Background Adenylate kinase 2 (AK2) is a phosphotransferase that catalyzes the reversible reaction 2ADP(GDP) ↔ ATP(GTP) + AMP and influences cellular energy homeostasis. However, the role of AK2 in regulating cell proliferation remains unclear because AK2 has been reported to be involved in either cell proliferation or cell apoptosis in different cell types of various organisms. Results This study reports AK2 promotion of cell proliferation using the lepidopteran insect Helicoverpa armigera and its epidermal cell line HaEpi as models. Western blot analysis indicates that AK2 constitutively expresses in various tissues during larval development. Immunocytochemistry analysis indicates that AK2 localizes in the mitochondria. The recombinant expressed AK2 in E. coli promotes cell growth and viability of HaEpi cell line by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. AK2 knockdown in larvae by RNA interference causes larval growth defects, including body weight decrease and development delay. AK2 knockdown in larvae also decreases the number of circulating haemocytes. The mechanism for such effects might be the suppression of gene transcription involved in insect development caused by AK2 knockdown. Conclusion These results show that AK2 regulates cell growth, viability, and proliferation in insect growth and development. PMID:23020757

  3. Cloning and characterization of a Drosophila serotonin receptor that activates adenylate cyclase.

    PubMed Central

    Witz, P; Amlaiky, N; Plassat, J L; Maroteaux, L; Borrelli, E; Hen, R

    1990-01-01

    Using a strategy based on nucleotide sequence homology between genes encoding receptors that interact with guanine nucleotide-binding proteins, we have isolated Drosophila genomic and cDNA clones encoding a functional serotonin receptor (5HT-dro receptor). This protein is expressed predominantly in Drosophila heads and exhibits highest homology with the human 5HT1A receptor. The predicted structure of the 5HT-dro receptor reveals two unusual features: (i) eight putative transmembrane domains instead of the expected seven and (ii) a Gly-Ser repeat that is a potential glycosaminoglycan attachment site. When stably introduced into mouse NIH 3T3 cells, the 5HT-dro receptor activates adenylate cyclase in response to serotonin and is inhibited by serotonin receptor antagonists such as dihydroergocryptine. The 5HT-dro receptor or closely related receptors might be responsible for the serotonin-sensitive cyclase that has been suggested to play a role in learning and modulation of circadian rhythm in a number of invertebrate systems. Images PMID:2174167

  4. The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

    PubMed Central

    Wang, Xianzhe; Maynard, Jennifer A.

    2015-01-01

    The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

  5. How adenylate cyclase choreographs the pas de deux of the receptors heteromerization dance.

    PubMed

    Woods, A S; Jackson, S N

    2013-05-15

    Our work suggests that heteromer formation, mainly involves linear motifs (LMs) found in disordered regions of proteins. Local disorder imparts plasticity to LMs. Most molecular recognition of proteins occurs between short linear segments, known as LMs. Interaction of short continuous epitopes is not constrained by sequence and has the advantage of resulting in interactions with micromolar affinities which suit transient, reversible complexes such as receptor heteromers. Electrostatic interactions between epitopes of the G-protein coupled receptors (GPCR) involved, are the key step in driving heteromer formation forward. The first step in heteromerization, involves phosphorylating Ser/Thr in an epitope containing a casein kinase 1/2-consensus site. Our data suggest that dopaminergic neurotransmission, through cAMP-dependent protein kinase A (PKA) slows down heteromerization. The negative charge, acquired by the phosphorylation of a Ser/Thr in a PKA consensus site in the Arg-rich epitope, affects the activity of the receptors involved in heteromerization by causing allosteric conformational changes, due to the repulsive effect generated by the negatively charged phosphate. In addition to modulating heteromerization, it affects the stability of the heteromers' interactions and their binding affinity. So here we have an instance where phosphorylation is not just an on/off switch, instead by weakening the noncovalent bond, heteromerization acts like a rheostat that controls the stability of the heteromer through activation or inhibition of adenylate cyclase by the neurotransmitter Dopamine depending on which Dopamine receptor it docks at. Published by Elsevier Ltd.

  6. Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin.

    PubMed

    Gonyar, Laura A; Gray, Mary C; Christianson, Gregory J; Mehrad, Borna; Hewlett, Erik L

    2017-06-01

    Pertussis (whooping cough), caused by Bordetella pertussis, is resurging in the United States and worldwide. Adenylate cyclase toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species. Copyright © 2017 American Society for Microbiology.

  7. The energy profiles of atomic conformational transition intermediates of adenylate kinase.

    PubMed

    Feng, Yaping; Yang, Lei; Kloczkowski, Andrzej; Jernigan, Robert L

    2009-11-15

    The elastic network interpolation (ENI) (Kim et al., Biophys J 2002;83:1620-1630) is a computationally efficient and physically realistic method to generate conformational transition intermediates between two forms of a given protein. However it can be asked whether these calculated conformations provide good representatives for these intermediates. In this study, we use ENI to generate conformational transition intermediates between the open form and the closed form of adenylate kinase (AK). Based on C(alpha)-only intermediates, we construct atomic intermediates by grafting all the atoms of known AK structures onto the C(alpha) atoms and then perform CHARMM energy minimization to remove steric conflicts and optimize these intermediate structures. We compare the energy profiles for all intermediates from both the CHARMM force-field and from knowledge-based energy functions. We find that the CHARMM energies can successfully capture the two energy minima representing the open AK and closed AK forms, while the energies computed from the knowledge-based energy functions can detect the local energy minimum representing the closed AK form and show some general features of the transition pathway with a somewhat similar energy profile as the CHARMM energies. The combinatorial extension structural alignment (Shindyalov et al., 1998;11:739-747) and the k-means clustering algorithm are then used to show that known PDB structures closely resemble computed intermediates along the transition pathway.

  8. Adenylate nucleotide levels and energy charge in Arthrobacter crystallopoietes during growth and starvation.

    PubMed

    Leps, W T; Ensign, J C

    1979-07-01

    The adenylate nucleotide concentrations, based on internal water space, were determined in cells of Arthrobacter crystallopoietes during growth and starvation and the energy charge of the cells was calculated. The energy charge of spherical cells rose during the first 10 h of growth, then remained nearly constant for as long as 20 h into the stationary phase. The energy charge of rod-shaped cells rose during the first 4 h of growth, then remained constant during subsequent growth and decreased in the stationary growth phase. Both spherical and rod-shaped cells excreted adenosine monophosphate but not adenosine triphosphate or adenosine diphosphate during starvation. The intracellular energy charge of spherical cells declined during the initial 10 h and then remained constant for 1 week of starvation at a value of 0.78. The intracellular energy charge of rod-shaped cells declined during the first 24 h of starvation, remained constant for the next 80 h, then decreased to a value of 0.73 after a total of 168 h starvation. Both cell forms remained more than 90% viable during this time. Addition of a carbon and energy source to starving cells resulted in an increase in the ATP concentration and as a result the energy charge increased to the smae levels as found during growth.

  9. Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate.

    PubMed

    Ribeiro, César; Esteves da Silva, Joaquim C G

    2008-09-01

    The inhibition mechanisms of the firefly luciferase (Luc) by the two major products of the reactions catalysed by Luc, oxyluciferin and dehydroluciferyl-adenylate (L-AMP), were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 microM oxyluciferin; 0.0025 to 1.25 microM L-AMP) has been measured in 50 mM Hepes buffer (pH=7.5), 10 nM Luc, 250 microM ATP and D-Luciferin (from 3.75 up to 120 microM). Nonlinear regression analysis with the appropriate kinetic models (Henri-Michaelis-Menten and William-Morrison equations) reveals that oxyluciferin is a competitive inhibitor of luciferase (Ki=0.50+/-0.03 microM) while L-AMP act as a tight-binding competitive inhibitor (Ki=3.8+/-0.7 nM). The Km values obtained both for oxyluciferin and L-AMP were 14.7+/-0.7 and 14.9+/-0.2 microM, respectively. L-AMP is a stronger inhibitor of Luc than oxyluciferin and the major responsible for the characteristic flash profile of in vitro Luc bioluminescence.

  10. Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

    PubMed Central

    Tlili, Mounira; Rouatbi, Sonia; Sriha, Badreddine; Ben Rhouma, Khémais; Sakly, Mohsen; Vaudry, David; Wurtz, Olivier; Tebourbi, Olfa

    2015-01-01

    The rate of atmospheric vanadium is constantly increasing due to fossil fuel combustion. This environmental pollution favours vanadium exposure in particular to its vanadate form, causing occupational bronchial asthma and bronchitis. Based on the well admitted bronchodilator properties of the pituitary adenylate cyclase-activating polypeptide (PACAP), we investigated the ability of this neuropeptide to reverse the vanadate-induced airway hyperresponsiveness in rats. Exposure to ammonium metavanadate aerosols (5 mg/m3/h) for 15 minutes induced 4 hours later an array of pathophysiological events, including increase of bronchial resistance and histological alterations, activation of proinflammatory alveolar macrophages, and increased oxidative stress status. Powerfully, PACAP inhalation (0.1 mM) for 10 minutes alleviated many of these deleterious effects as demonstrated by a decrease of bronchial resistance and histological restoration. PACAP reduced the level of expression of mRNA encoding inflammatory chemokines (MIP-1α, MIP-2, and KC) and cytokines (IL-1α and TNF-α) in alveolar macrophages and improved the antioxidant status. PACAP reverses the vanadate-induced airway hyperresponsiveness not only through its bronchodilator activity but also by counteracting the proinflammatory and prooxidative effects of the metal. Then, the development of stable analogs of PACAP could represent a promising therapeutic alternative for the treatment of inflammatory respiratory disorders. PMID:26199679

  11. Cellular localization of pituitary adenylate cyclase-activating peptide (PACAP) following traumatic brain injury in humans.

    PubMed

    van Landeghem, Frank K H; Weiss, Thorsten; Oehmichen, Manfred; von Deimling, Andreas

    2007-06-01

    The pituitary adenylate cyclase-activating peptide (PACAP) is involved in many processes of the developing and mature central nervous system, such as proliferation, differentiation, apoptosis, neurotransmission, inflammation and neuroprotection. Alternative posttranslational processing of PACAP results in two biologically active, amidated 27- and 38-amino acid peptides termed PACAP27 and PACAP38. In the present study, we examined whether traumatic brain injury (TBI) affects cellular immunopositivity for PACAP27 and PACAP38. Patients (n = 55) were classified into three groups dependent on their survival time (under 24 h, between 24 h and 7 days and between 7 days and 99 days postinjury). PACAP27 and PACAP38 were expressed by neurons and glial cells in normal human neocortex (n = 10). Following TBI, the total number of PACAP27- and PACAP38-positive cells was significantly decreased for a prolonged survival period within the traumatized neocortex. In the pericontusional cortex, the number of cells expressing PACAP27 and PACAP38 was significantly increased at all survival times examined. Triple immunofluorescence examinations revealed a significant increase in the absolute numbers of GFAP-positive reactive astrocytes as well as a decrease in the CNP-positive oligodendrocytes, each coexpressing PACAP27 or PACAP38 in the contusional and pericontusional cortex. We hypothesize that the increase of glial PACAP immunoreactivity may be interpreted as part of a complex endogenous neuroprotective response in the pericontusional regions, but the precise role of PACAP following TBI is yet to be determined.

  12. The Rhizobium etli cyaC product: characterization of a novel adenylate cyclase class.

    PubMed

    Téllez-Sosa, Juan; Soberón, Nora; Vega-Segura, Alicia; Torres-Márquez, María E; Cevallos, Miguel A

    2002-07-01

    Adenylate cyclases (ACs) catalyze the formation of 3',5'-cyclic AMP (cAMP) from ATP. A novel AC-encoding gene, cyaC, was isolated from Rhizobium etli by phenotypic complementation of an Escherichia coli cya mutant. The functionality of the cyaC gene was corroborated by its ability to restore cAMP accumulation in an E. coli cya mutant. Further, overexpression of a malE::cyaC fusion protein allowed the detection of significant AC activity levels in cell extracts of an E. coli cya mutant. CyaC is unrelated to any known AC or to any other protein exhibiting a currently known function. Thus, CyaC represents the first member of a novel class of ACs (class VI). Hypothetical genes of unknown function similar to cyaC have been identified in the genomes of the related bacterial species Mesorhizobium loti, Sinorhizobium meliloti, and Agrobacterium tumefaciens. The cyaC gene is cotranscribed with a gene similar to ohr of Xanthomonas campestris and is expressed only in the presence of organic hydroperoxides. The physiological performance of an R. etli cyaC mutant was indistinguishable from that of the wild-type parent strain both under free-living conditions and during symbiosis.

  13. The Rhizobium etli cyaC Product: Characterization of a Novel Adenylate Cyclase Class

    PubMed Central

    Téllez-Sosa, Juan; Soberón, Nora; Vega-Segura, Alicia; Torres-Márquez, María E.; Cevallos, Miguel A.

    2002-01-01

    Adenylate cyclases (ACs) catalyze the formation of 3′,5′-cyclic AMP (cAMP) from ATP. A novel AC-encoding gene, cyaC, was isolated from Rhizobium etli by phenotypic complementation of an Escherichia coli cya mutant. The functionality of the cyaC gene was corroborated by its ability to restore cAMP accumulation in an E. coli cya mutant. Further, overexpression of a malE::cyaC fusion protein allowed the detection of significant AC activity levels in cell extracts of an E. coli cya mutant. CyaC is unrelated to any known AC or to any other protein exhibiting a currently known function. Thus, CyaC represents the first member of a novel class of ACs (class VI). Hypothetical genes of unknown function similar to cyaC have been identified in the genomes of the related bacterial species Mesorhizobium loti, Sinorhizobium meliloti, and Agrobacterium tumefaciens. The cyaC gene is cotranscribed with a gene similar to ohr of Xanthomonas campestris and is expressed only in the presence of organic hydroperoxides. The physiological performance of an R. etli cyaC mutant was indistinguishable from that of the wild-type parent strain both under free-living conditions and during symbiosis. PMID:12057950

  14. Variation in adenylate energy charge and phosphoadenylate pool size in estuarine organisms after an oil spill

    SciTech Connect

    Shafer, T.H.; Hackney, C.T.

    1987-05-01

    Adenylate energy charge (AEC) is the proportion of the total phosphoadenylate pool charged with high-energy bonds. AEC values vary between zero and one by definition. Since AEC can be measured in any organism, decreases might be a universal measure of sublethal environmental stress. In some organisms which maintain high AEC while withstanding natural or anthropogenic stress, the absolute concentration of ATP and the total phosphoadenylate pool (TPP) decrease proportionally. However, in certain organisms the TPP shows dramatic natural fluctuations unrelated to pollution or stress. On 28 June 1983, a tanker spilled approximately 42,000 gallons of number6 diesel oil in the Cape Fear River, North Carolina, USA. Oil covered the tidal marshes on the east side of the river and provided an opportunity to determine if either the AEC or TPP in a variety of organisms would respond to this stress. Five test species were examined as long as one year after the spill. AEC and TPP values of the organisms were compared between contaminated and uncontaminated sites at all seasons. This is the first investigation to monitor AEC in a number of taxonomically distinct estuarine species during an extended period after an oil spill.

  15. Adenylate Cyclase Type III Is Not a Ubiquitous Marker for All Primary Cilia during Development

    PubMed Central

    Antal, Maria Cristina; Bénardais, Karelle; Samama, Brigitte; Auger, Cyril; Schini-Kerth, Valérie; Ghandour, Said; Boehm, Nelly

    2017-01-01

    Adenylate cyclase type III (AC3) is localized in plasma membrane of neuronal primary cilium and can be used as a marker of this cilium. AC3 has also been detected in some other primary cilia such as those of fibroblasts, synoviocytes or astrocytes. Despite the presence of a cilium in almost all cell types, we show that AC3 is not a common marker of all primary cilia of different human and mouse tissues during development. In peripheral organs, AC3 is present mainly in primary cilia in cells of the mesenchymal lineage (fibroblasts, chondroblasts, osteoblasts-osteocytes, odontoblasts, muscle cells and endothelial cells). In epithelia, the apical cilium of renal and pancreatic tubules and of ductal plate in liver is AC3-negative whereas the cilium of basal cells of stratified epithelia is AC3-positive. Using fibroblasts cell culture, we show that AC3 appears at the plasma membrane of the primary cilium as soon as this organelle develops. The functional significance of AC3 localization at the cilium membrane in some cells but not others has to be investigated in relationship with cell physiology and expression at the cilium plasma membrane of specific upstream receptors. PMID:28122017

  16. Energy landscape and multiroute folding of topologically complex proteins adenylate kinase and 2ouf-knot

    PubMed Central

    Li, Wenfei; Terakawa, Tsuyoshi; Wang, Wei; Takada, Shoji

    2012-01-01

    While fast folding of small proteins has been relatively well characterized by experiments and theories, much less is known for slow folding of larger proteins, for which recent experiments suggested quite complex and rich folding behaviors. Here, we address how the energy landscape theory can be applied to these slow folding reactions. Combining the perfect-funnel approximation with a multiscale method, we first extended our previous atomic-interaction based coarse grained (AICG) model to take into account local flexibility of protein molecules. Using this model, we then investigated the energy landscapes and folding routes of two proteins with complex topologies: a multidomain protein adenylate kinase (AKE) and a knotted protein 2ouf-knot. In the AKE folding, consistent with experimental results, the kinetic free energy surface showed several substates between the fully unfolded and native states. We characterized the structural features of these substates and transitions among them, finding temperature-dependent multiroute folding. For protein 2ouf-knot, we found that the improved atomic-interaction based coarse-grained model can spontaneously tie a knot and fold the protein with a probability up to 96%. The computed folding rate of the knotted protein was much slower than that of its unknotted counterpart, in agreement with experimental findings. Similar to the AKE case, the 2ouf-knot folding exhibited several substates and transitions among them. Interestingly, we found a dead-end substate that lacks the knot, thus suggesting backtracking mechanisms. PMID:22753508

  17. Inhibition of lipolysis by agents acting via adenylate cyclase in fat cells from infants and adults.

    PubMed

    Marcus, C; Sonnenfeld, T; Karpe, B; Bolme, P; Arner, P

    1989-09-01

    The in vitro lipolytic effect of catecholamines is poor during infancy because of enhanced alpha 2-adrenoceptor activity. The mechanisms behind this were investigated in isolated fat cells obtained from 1- to 4-mo-old infants and from adults. The cells were incubated with agents that inhibit lipolysis through distinct receptors coupled to adenylate cyclase via the inhibitory GTP binding coupling protein, Gi. The sensitivity to the alpha 2-adrenoceptor agonist clonidine was 14 times higher in the infant group as compared to the adults, whereas that to an adenosine analogue was 14 times lower. The sensitivities to prostaglandin E2 and nicotinic acid were similar in both age groups. Preincubation of the adipocytes with pertussis toxin abolished the antilipolytic effects of all agents. The density of alpha 2-adrenoceptor binding sites determined with [3H]yohimbine was increased by about 25% in the infants. In conclusion, the antilipolytic sensitivity of adenosine and alpha 2-adrenoceptors develops separately and may play different roles in the regulation of lipolysis in man. Furthermore, the enhanced alpha 2-adrenoceptor sensitivity during infancy seems at least in part to be due to an increase in the number of receptors.

  18. Posttraumatic administration of pituitary adenylate cyclase activating polypeptide in central fluid percussion injury in rats.

    PubMed

    Kövesdi, Erzsébet; Tamás, Andrea; Reglodi, Dóra; Farkas, Orsolya; Pál, József; Tóth, Gábor; Bukovics, Péter; Dóczi, Tamás; Büki, András

    2008-04-01

    Several in vitro and in vivo experiments have demonstrated the neuroprotective effects of pituitary adenylate cyclase activating polypeptide (PACAP) in focal cerebral ischemia, Parkinson's disease and traumatic brain injury (TBI). The aim of the present study was to analyze the effect of PACAP administration on diffuse axonal injury (DAI), an important contributor to morbidity and mortality associated with TBI, in a central fluid percussion (CFP) model of TBI. Rats were subjected to moderate (2 Atm) CFP injury. Thirty min after injury, 100 microg PACAP was administered intracerebroventricularly. DAI was assessed by immunohistochemical detection of beta-amyloid precursor protein, indicating impaired axoplasmic transport, and RMO-14 antibody, representing foci of cytoskeletal alterations (neurofilament compaction), both considered classical markers of axonal damage. Analysis of damaged, immunoreactive axonal profiles revealed significant axonal protection in the PACAP-treated versus vehicle-treated animals in the corticospinal tract, as far as traumatically induced disturbance of axoplasmic transport and cytoskeletal alteration were considered. Similarly to our former observations in an impact acceleration model of diffuse TBI, the present study demonstrated that PACAP also inhibits DAI in the CFP injury model. The finding indicates that PACAP and derivates can be considered potential candidates for further experimental studies, or purportedly for clinical trials in the therapy of TBI.

  19. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3

    PubMed Central

    Osicka, Radim; Osickova, Adriana; Hasan, Shakir; Bumba, Ladislav; Cerny, Jiri; Sebo, Peter

    2015-01-01

    Integrins are heterodimeric cell surface adhesion and signaling receptors that are essential for metazoan existence. Some integrins contain an I-domain that is a major ligand binding site. The ligands preferentially engage the active forms of the integrins and trigger signaling cascades that alter numerous cell functions. Here we found that the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis, preferentially binds an inactive form of the integrin complement receptor 3 (CR3), using a site outside of its I-domain. CyaA binding did not trigger downstream signaling of CR3 in human monocytes and CyaA-catalyzed elevation of cAMP effectively blocked CR3 signaling initiated by a natural ligand. This unprecedented type of integrin-ligand interaction distinguishes CyaA from all other known ligands of the I-domain-containing integrins and provides a mechanistic insight into the previously observed central role of CyaA in the pathogenesis of B. pertussis. DOI: http://dx.doi.org/10.7554/eLife.10766.001 PMID:26650353

  20. Pituitary adenylate cyclase-activating polypeptide is reduced in Alzheimer disease.

    PubMed

    Han, Pengcheng; Liang, Winnie; Baxter, Leslie C; Yin, Junxiang; Tang, Zhiwei; Beach, Thomas G; Caselli, Richard J; Reiman, Eric M; Shi, Jiong

    2014-05-13

    There is growing evidence that pituitary adenylate cyclase-activating polypeptide (PACAP) is associated with Alzheimer disease (AD) pathology in animal models, but human studies are needed. We studied the brains of patients with pathologically confirmed late-onset AD and age-matched cognitively normal (CN) subjects to investigate the expression of PACAP messenger RNA (34 AD and 14 CN) and protein (12 AD and 11 CN) in a case-control study. We report that PACAP levels are reduced in multiple brain regions, including the entorhinal cortex, the middle temporal gyrus, the superior frontal gyrus, and the primary visual cortex. This reduction is correlated with higher amyloid burden (CERAD plaque density) in the entorhinal cortex and superior frontal gyrus but not in the primary visual cortex, a region spared in most cases of AD. PACAP expression is lower in advanced Braak stages (V and VI) than in moderate stages (III and IV). Increased PACAP levels are associated with decreased scores on the Dementia Rating Scale, a global cognitive measure. Finally, CSF levels paralleled brain levels in AD but not in Parkinson dementia or frontotemporal dementia brains. The close relationship between PACAP reduction and the severity of AD pathology suggests that downregulation of PACAP may contribute to AD pathogenesis.

  1. Adenylate Cyclase Type III Is Not a Ubiquitous Marker for All Primary Cilia during Development.

    PubMed

    Antal, Maria Cristina; Bénardais, Karelle; Samama, Brigitte; Auger, Cyril; Schini-Kerth, Valérie; Ghandour, Said; Boehm, Nelly

    2017-01-01

    Adenylate cyclase type III (AC3) is localized in plasma membrane of neuronal primary cilium and can be used as a marker of this cilium. AC3 has also been detected in some other primary cilia such as those of fibroblasts, synoviocytes or astrocytes. Despite the presence of a cilium in almost all cell types, we show that AC3 is not a common marker of all primary cilia of different human and mouse tissues during development. In peripheral organs, AC3 is present mainly in primary cilia in cells of the mesenchymal lineage (fibroblasts, chondroblasts, osteoblasts-osteocytes, odontoblasts, muscle cells and endothelial cells). In epithelia, the apical cilium of renal and pancreatic tubules and of ductal plate in liver is AC3-negative whereas the cilium of basal cells of stratified epithelia is AC3-positive. Using fibroblasts cell culture, we show that AC3 appears at the plasma membrane of the primary cilium as soon as this organelle develops. The functional significance of AC3 localization at the cilium membrane in some cells but not others has to be investigated in relationship with cell physiology and expression at the cilium plasma membrane of specific upstream receptors.

  2. [Structure, localization and physiologic role of pituitary adenylate cyclase activating polypeptide (PACAP)].

    PubMed

    Vincze, E; Köves, K

    2001-03-11

    PACAP was isolated on the basis of its ability to stimulate adenylate cyclase in primary anterior pituitary cell culture from ovine hypothalami by Miyata et al. in 1989. This peptide is structurally related to the secretin family and shows a 67% sequence homology with vasoactive intestinal polypeptide (VIP). The amino acid sequence of PACAP has been highly preserved during the evolution that may be connected with its important physiological role. Similar to other "brain-gut peptides" PACAP is localized not only in the central but in the peripheral nervous system and in non-neural tissues as well. In addition to its hypophysiotropic effects in the hypothalamo-hypophysial system PACAP exerts its effects on water-salt balance, cardiovascular functions, gastrointestinal motility and secretion and also on the regulation of reproductive functions. PACAP has a role in certain neuro-immuno-endocrine processes, in the differentiation of the nervous system, and it has neuroprotective effects in the case of ischaemia and various toxic agents. Locally PACAP takes its effects as an auto- and paracrine hormone, a neurotransmitter or a neuromodulator in different organs. Besides VIP, PACAP plays an important role in the function of the photo-neuro-endocrine system.

  3. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Is Involved in Adult Mouse Hippocampal Neurogenesis After Stroke.

    PubMed

    Matsumoto, Minako; Nakamachi, Tomoya; Watanabe, Jun; Sugiyama, Koichi; Ohtaki, Hirokazu; Murai, Norimitsu; Sasaki, Shun; Xu, Zhifang; Hashimoto, Hitoshi; Seki, Tamotsu; Miyazaki, Akira; Shioda, Seiji

    2016-06-01

    In the subgranular zone (SGZ) of the hippocampus, neurogenesis persists throughout life and is upregulated following ischemia. Accumulating evidence suggests that enhanced neurogenesis stimulated by ischemic injury contributes to recovery after stroke. However, the mechanisms underlying the upregulation of neurogenesis are unclear. We have demonstrated that a neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), exerts a wide range of effects on neural stem cells (NSCs) during neural development. Here, we examined the effects of endogenous and exogenous PACAP in adult NSCs of the SGZ. Immunostaining showed expression of the PACAP receptor PAC1R in nestin-positive NSCs of adult naive mice. PACAP injection into the lateral ventricle increased bromodeoxyuridine (BrdU)-positive proliferative cells in the SGZ. These data suggest that PACAP promoted the proliferation of NSCs. In global ischemia model mice, the number of BrdU-positive cells was increased in wild-type mice but not in PACAP heterozygous knockout mice. The BrdU-positive cells that increased in number after ischemia were immunopositive for SOX2, a marker of NSCs, and differentiated into NeuN-positive mature neurons at 4 weeks after ischemia. These findings suggest that PACAP contributes to the proliferation of NSCs and may be associated with recovery after brain injury.

  4. Texture Analysis of Poly-Adenylated mRNA Staining Following Global Brain Ischemia and Reperfusion

    PubMed Central

    Szymanski, Jeffrey J.; Jamison, Jill T.; DeGracia, Donald J.

    2011-01-01

    Texture analysis provides a means to quantify complex changes in microscope images. We previously showed that cytoplasmic poly-adenylated mRNAs form mRNA granules in post-ischemic neurons and that these granules correlated with protein synthesis inhibition and hence cell death. Here we utilized the texture analysis software MaZda to quantify mRNA granules in photomicrographs of the pyramidal cell layer of rat hippocampal region CA3 around 1 hour of reperfusion after 10 min of normothermic global cerebral ischemia. At 1 hour reperfusion, we observed variations in the texture of mRNA granules amongst samples that were readily quantified by texture analysis. Individual sample variation was consistent with the interpretation that animal-to-animal variations in mRNA granules reflected the time-course of mRNA granule formation. We also used texture analysis to quantify the effect of cycloheximide, given either before or after brain ischemia, on mRNA granules. If administered before ischemia, cycloheximide inhibited mRNA granule formation, but if administered after ischemia did not prevent mRNA granulation, indicating mRNA granule formation is dependent on dissociation of polysomes. We conclude that texture analysis is an effective means for quantifying the complex morphological changes induced in neurons by brain ischemia and reperfusion. PMID:21477879

  5. Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase

    PubMed Central

    Abdo, Zaid; Byers, Sara Overstreet; Kriebel, Patrick; Rothrock, Michael J.

    2016-01-01

    Abstract Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition. PMID:27035032

  6. Effects of forskolin on cerebral blood flow: implications for a role of adenylate cyclase

    SciTech Connect

    Wysham, D.G.; Brotherton, A.F.; Heistad, D.D.

    1986-11-01

    We have studied cerebral vascular effects of forskolin, a drug which stimulates adenylate cyclase and potentiates dilator effects of adenosine in other vascular beds. Our goals were to determine whether forskolin is a cerebral vasodilator and whether it potentiates cerebral vasodilator responses to adenosine. We measured cerebral blood flow with microspheres in anesthetized rabbits. Forskolin (10 micrograms/kg per min) increased blood flow (ml/min per 100 gm) from 39 +/- 5 (mean +/- S.E.) to 56 +/- 9 (p less than 0.05) in cerebrum, and increased flow to myocardium and kidney despite a decrease in mean arterial pressure. Forskolin did not alter cerebral oxygen consumption, which indicates that the increase in cerebral blood flow is a direct vasodilator effect and is not secondary to increased metabolism. We also examined effects of forskolin on the response to infusion of adenosine. Cerebral blood flow was measured during infusion of 1-5 microM/min adenosine into one internal carotid artery, under control conditions and during infusion of forskolin at 3 micrograms/kg per min i.v. Adenosine alone increased ipsilateral cerebral blood flow from 32 +/- 3 to 45 +/- 5 (p less than 0.05). Responses to adenosine were not augmented during infusion of forskolin. We conclude that forskolin is a direct cerebral vasodilator and forskolin does not potentiate cerebral vasodilator responses to adenosine.

  7. Cytochemical localization of adenylate cyclase activity in heart tissue with cerium.

    PubMed

    Schulze, W; Will-Shahab, L; Küttner, I

    1986-01-01

    Adenylate cyclase (AC) activity showed a doses depending inactivation of the basal activity and of the sodium fluoride stimulation by cerium in homogenates of unfixed and fixed guinea pig hearts. The isoproterenol and guanine nucleotide stimulation was not more than two times of the basal activity in glutaraldehyde-prefixed heart homogenates in the presence of 2 mmol/l CeCl3. The inactivation of the AC (activity) by cerium was less than in the presence of lead. Test tube experiments showed no differences in the precipitation of imidodiphosphate in comparison with inorganic phosphate. The substrate AMP-PNP was not spontaneously hydrolysed by 2 mmol/l CeCl3. Ultrastructural analysis of cytochemical incubation of glutaraldehyde-fixed slices and small pieces of guinea pig heart tissue showed fine-amorphous precipitations of reaction products localized along the plasma membrane of the sarcolemma, the nexuses of the intercalated discs and the T-tubule membranes. No precipitates were found neither on the junctional nor on other SR membranes. Nonspecific coarse and clumped precipitates have been detected in the intercellular space on components of the basal membranes. It was not able to demonstrate cytochemically stimulation of AC by hormones or by sodium fluoride. The localization of the basal AC activity in heart tissue seems to be better with cerium as capture agent than with lead. However, differences in the localization of the AC activity in heart tissue were not observed.

  8. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  9. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  10. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  11. Engineering enzymes.

    PubMed

    Dutton, P Leslie; Moser, Christopher C

    2011-01-01

    Fundamental research into bioinorganic catalysis of the kind presented at this Faraday Discussion has the potential to turn inspiration drawn from impressive natural energy and chemical transformations into artificial catalyst constructions useful to mankind. Creating bio-inspired artificial constructions requires a level of understanding well beyond simple description of structures and mechanisms of natural enzymes. To be useful, such description must be augmented by a practical sense of structural and energetic engineering tolerances of the mechanism. Significant barriers to achieving an engineering understanding of enzyme mechanisms arise from natural protein complexity. In certain cases we can surmount these barriers to understanding, such as natural electron tunneling, coupling of electron tunneling to light capture and proton exchange as well as simpler bond breaking redox catalysis. Hope for similar solutions of more complex bioinorganic enzymes is indicated in several papers presented in this Discussion. Armed with an engineering understanding of mechanism, the current serious frustrations to successful creation of functional artificial proteins that are rooted in protein complexity can fall away. Here we discuss the genetic and biological roots of protein complexity and show how to dodge and minimize the effects of complexity. In the best-understood cases, artificial enzymes can be designed from scratch using the simplest of protein scaffolds.

  12. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  13. Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2.

    PubMed

    Lange, Stephan; Auerbach, Daniel; McLoughlin, Patricia; Perriard, Evelyne; Schäfer, Beat W; Perriard, Jean-Claude; Ehler, Elisabeth

    2002-12-15

    During sarcomere contraction skeletal and cardiac muscle cells consume large amounts of energy. To satisfy this demand, metabolic enzymes are associated with distinct regions of the sarcomeres in the I-band and in the M-band, where they help to maintain high local concentrations of ATP. To date, the mechanism by which metabolic enzymes are coupled to the sarcomere has not been elucidated. Here, we show that the four and a half LIM-only protein DRAL/FHL-2 mediates targeting of the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase by interaction with the elastic filament protein titin in cardiomyocytes. Using yeast two-hybrid assays, colocalisation experiments, co-immunoprecipitation and protein pull-down assays, we show that DRAL/FHL-2 is bound to two distinct sites on titin. One binding site is situated in the N2B region, a cardiac-specific insertion in the I-band part of titin, and the other is located in the is2 region of M-band titin. We also show that DRAL/FHL-2 binds to the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase and might target these enzymes to the N2B and is2 regions in titin. We propose that DRAL/FHL-2 acts as a specific adaptor protein to couple metabolic enzymes to sites of high energy consumption in the cardiac sarcomere.

  14. Leishmania donovani tyrosyl-tRNA synthetase structure in complex with a tyrosyl adenylate analog and comparisons with human and protozoan counterparts.

    PubMed

    Barros-Álvarez, Ximena; Kerchner, Keshia M; Koh, Cho Yeow; Turley, Stewart; Pardon, Els; Steyaert, Jan; Ranade, Ranae M; Gillespie, J Robert; Zhang, Zhongsheng; Verlinde, Christophe L M J; Fan, Erkang; Buckner, Frederick S; Hol, Wim G J

    2017-07-01

    The crystal structure of Leishmania donovani tyrosyl-tRNA synthetase (LdTyrRS) in complex with a nanobody and the tyrosyl adenylate analog TyrSA was determined at 2.75 Å resolution. Nanobodies are the variable domains of camelid heavy chain-only antibodies. The nanobody makes numerous crystal contacts and in addition reduces the flexibility of a loop of LdTyrRS. TyrSA is engaged in many interactions with active site residues occupying the tyrosine and adenine binding pockets. The LdTyrRS polypeptide chain consists of two pseudo-monomers, each consisting of two domains. Comparing the two independent chains in the asymmetric unit reveals that the two pseudo-monomers of LdTyrRS can bend with respect to each other essentially as rigid bodies. This flexibility might be useful in the positioning of tRNA for catalysis since both pseudo-monomers in the LdTyrRS chain are needed for charging tRNA(Tyr). An "extra pocket" (EP) appears to be present near the adenine binding region of LdTyrRS. Since this pocket is absent in the two human homologous enzymes, the EP provides interesting opportunities for obtaining selective drugs for treating infections caused by L. donovani, a unicellular parasite causing visceral leishmaniasis, or kala azar, which claims 20,000 to 30,000 deaths per year. Sequence and structural comparisons indicate that the EP is a characteristic which also occurs in the active site of several other important pathogenic protozoa. Therefore, the structure of LdTyrRS could inspire the design of compounds useful for treating several different parasitic diseases. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

    PubMed

    Fernández, Silvina; Córdoba, Mariana

    2017-04-01

    Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC.

  16. Structural characterization by nuclear magnetic resonance spectroscopy of a genetically engineered high-affinity calmodulin-binding peptide derived from Bordetella pertussis adenylate cyclase.

    PubMed

    Munier, H; Bouhss, A; Gilles, A M; Palibroda, N; Bârzu, O; Mispelter, J; Craescu, C T

    1995-07-10

    This paper reports the solution conformation of a peptide (P196-267) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. P196-267 corresponding to the protein fragment situated between amino acid residues 196-267 was overproduced by a recombinant Escherichia coli strain. Its affinity for calmodulin is only one order of magnitude lower (Kd = 2.4 nM) than that of the whole bacterial enzyme (Kd = 0.2 nM). The proton resonances of the NMR spectra of P196-267 were assigned using homonuclear two-dimensional techniques (double-quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser enhancement spectroscopy) and a standard assignment procedure. Analysis of the nuclear Overhauser effect connectivities and the secondary shift distribution of C alpha protons along the sequence allowed us to identify the elements of regular secondary structure. The peptide is flexible in solution, being in equilibrium between random coil and helical structures. Two segments of 11 amino acids (situated between V215 and A225) and 15 amino acids (situated between L233 and A247) populate in a significant proportion the helix conformational state. The two helices can be considerably stabilized in a mixed solvent, trifluoroethanol/water (30/70), suggesting that the corresponding fragment in the intact protein assumes a similar secondary conformation. No elements of tertiary structure organization were detected by the present experiments. The conformational properties of the isolated calmodulin target fragment are discussed in relation with the available NMR and X-ray data on various peptides complexed to calmodulin.

  17. Classification of the adenylation and acyl-transferase activity of NRPS and PKS systems using ensembles of substrate specific hidden Markov models.

    PubMed

    Khayatt, Barzan I; Overmars, Lex; Siezen, Roland J; Francke, Christof

    2013-01-01

    There is a growing interest in the Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) of microbes, fungi and plants because they can produce bioactive peptides such as antibiotics. The ability to identify the substrate specificity of the enzyme's adenylation (A) and acyl-transferase (AT) domains is essential to rationally deduce or engineer new products. We here report on a Hidden Markov Model (HMM)-based ensemble method to predict the substrate specificity at high quality. We collected a new reference set of experimentally validated sequences. An initial classification based on alignment and Neighbor Joining was performed in line with most of the previously published prediction methods. We then created and tested single substrate specific HMMs and found that their use improved the correct identification significantly for A as well as for AT domains. A major advantage of the use of HMMs is that it abolishes the dependency on multiple sequence alignment and residue selection that is hampering the alignment-based clustering methods. Using our models we obtained a high prediction quality for the substrate specificity of the A domains similar to two recently published tools that make use of HMMs or Support Vector Machines (NRPSsp and NRPS predictor2, respectively). Moreover, replacement of the single substrate specific HMMs by ensembles of models caused a clear increase in prediction quality. We argue that the superiority of the ensemble over the single model is caused by the way substrate specificity evolves for the studied systems. It is likely that this also holds true for other protein domains. The ensemble predictor has been implemented in a simple web-based tool that is available at http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/.

  18. Targeted mutations that ablate either the adenylate cyclase or hemolysin function of the bifunctional cyaA toxin of Bordetella pertussis abolish virulence.

    PubMed Central

    Gross, M K; Au, D C; Smith, A L; Storm, D R

    1992-01-01

    Bordetella pertussis, the causative agent of whooping cough, secretes several toxins implicated in this disease. One of these putative virulence factors is the adenylate cyclase (AC) toxin that elevates intracellular cAMP in eukaryotic cells to cytotoxic levels. This toxin is a bifunctional protein comprising both AC and hemolysin (HLY) enzymatic domains. The gene encoding the AC toxin (cyaA) is expressed as part of an operon that includes genes required for secretion or activation of the toxin. Because of this genetic organization, it is difficult to create B. pertussis mutants of cyaA that are ablations of a single enzyme function by conventional means, such as transposon mutagenesis. Therefore, to clarify the role of individual toxin functions in the virulence of B. pertussis, we have used site-directed or deletion mutagenesis and genetic recombination to specifically target the cyaA gene of B. pertussis to produce mutants that lack only the AC or HLY activity of this toxin. A point mutant of B. pertussis with abolished AC catalytic activity was greater than 1000 times less pathogenic to newborn mice than wild-type bacteria, directly demonstrating the importance of the AC toxin in pertussis virulence. Similarly, an in-frame deletion mutant of B. pertussis that lacks HLY is equally avirulent, supporting observations that the HLY domain plays a critical role in AC toxin entry into cells. Furthermore, the genetically inactivated AC toxin produced by the point mutant is antigenically similar to the native toxin, suggesting that this strain may be useful in the development of pertussis component vaccines. Images PMID:1594590

  19. Chronic ghrelin treatment reduced photophobia and anxiety-like behaviors in nitroglycerin- induced migraine: role of pituitary adenylate cyclase-activating polypeptide.

    PubMed

    Farajdokht, Fereshteh; Babri, Shirin; Karimi, Pouran; Alipour, Mohammad Reza; Bughchechi, Ramin; Mohaddes, Gisou

    2017-03-01

    Chronic migraine is a debilitating disorder that has a significant impact on patients and society. Nearly all migraineurs frequently reported light sensitivity during a headache attack. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the activation of trigeminal system and migraine pain. To identify the effect of chronic ghrelin treatment on endogenous PACAP and associated symptoms of migraine, an experimental chronic migraine model was induced by intermittent intraperitoneal (i.p) injection of nitroglycerin (NTG). Photophobia and anxiety-like behaviors were determined in the modified elevated plus maze on days 2, 4, 6, 8, and 10 and in the light/dark box on days 3, 5, 7, 9, and 11. Blood levels of PACAP and cortisol were assessed by enzyme-linked immunosorbent (ELISA) kits. Chronic injection of NTG evoked photophobia and anxiety-like behaviors and treatment with ghrelin (150 μg/kg) for 11 days effectively attenuated photophobia and anxiety-like behaviors in the both paradigms. We further found that NTG increased the blood levels of PACAP and cortisol, which was significantly reduced by ghrelin treatment. Additionally, staining with Hematoxylin and Eosin (H&E) revealed that ghrelin reduced NTG-induced increase in the number of satellite glial cells in the trigeminal ganglion. Furthermore, for the first time we showed that repeated administrations of NTG increased white blood cell (WBC) counts and mean platelet volume (MPV), and decreased platelet counts. These results indicated that ghrelin decreased migraine associated symptoms possibly through attenuating endogenous PACAP and cortisol levels. Therefore, ghrelin may hold therapeutic potentialities in managing the chronic migraine.

  20. Mechanism of adenylate kinase. Demonstration of a functional relationship between aspartate 93 and Mg2+ by site-directed mutagenesis and proton, phosphorus-31, and magnesium-25 NMR.

    PubMed

    Yan, H G; Tsai, M D

    1991-06-04

    Earlier magnetic resonance studies suggested no direct interaction between Mg2+ ions and adenylate kinase (AK) in the AK.MgATP (adenosine 5'-triphosphate) complex. However, recent NMR studies concluded that the carboxylate of aspartate 119 accepts a hydrogen bond from a water ligand of the bound Mg2+ ion in the muscle AK.MgATP complex [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. On the other hand, in the 2.6-A crystal structure of the yeast AK.MgAP5A [P1,P5-bis(5'-adenosyl)pentaphosphate] complex, the Mg2+ ion is in proximity to aspartate 93 [Egner, U., Tomasselli, A.G., & Schulz, G.E. (1987) J. Mol. Biol. 195, 649-658]. Substitution of Asp-93 with alanine resulted in no change in dissociation constants, 4-fold increases in Km, and a 650-fold decrease in kcat. Notable changes have been observed in the chemical shifts of the aromatic protons of histidine 36 and a few other aromatic residues. However, the results of detailed analyses of the free enzymes and the AK.MgAP5A complexes by one- and two-dimensional NMR suggested that the changes are due to localized perturbations. Thus it is concluded that Asp-93 stabilizes the transition state by ca. 3.9 kcal/mol. The next question is how. Since proton NMR results indicated that binding of Mg2+ to the AK.AP5A complex induces some changes in the proton NMR signals of WT but not those of D93A, the functional role of Asp-93 should be in binding to Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D/sub 2/ receptor

    SciTech Connect

    Borgundvaag, B.; George, S.R.

    1985-07-29

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of (/sup 3/H)-ATP to (/sup 3/H)-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC/sub 50/ values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC/sub 50/ values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D/sub 2/ dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table.

  2. D-1 dopaminergic and beta-adrenergic stimulation of adenylate cyclase in a clone derived from the human astrocytoma cell line G-CCM.

    PubMed

    Balmforth, A J; Ball, S G; Freshney, R I; Graham, D I; McNamee, H B; Vaughan, P F

    1986-09-01

    Clones have been isolated from the human astrocytoma cell line G-CCM. Homogenates of clone D384 contain an adenylate cyclase that is stimulated by 3,4-dihydroxyphenylethylamine (dopamine), noradrenaline, and isoprenaline with Ka apparent values of 4, 56, and 2.7 microM, respectively. The Ka apparent value for dopamine was increased by the D-1 antagonist cis-flupenthixol, 25 and 100 nM, to 23 and 190 microM, respectively, but was unaffected by propranolol (1 microM). Noradrenaline stimulation of adenylate cyclase was only partially inhibited by either propranolol (10 microM) or cis-flupenthixol (1 microM). Propranolol (10 microM), but not cis-flupenthixol (1 microM), prevented stimulation by isoprenaline. The stimulation of adenylate cyclase by dopamine and noradrenaline remained unchanged in the presence of phentolamine (1 microM) and sulpiride (1 microM). These results suggest that clone D384 contains both D-1 dopaminergic and beta-adrenergic receptors coupled to adenylate cyclase. Dopamine stimulates D384 adenylate cyclase through D-1 receptors, isoprenaline via beta-receptors, and noradrenaline through both receptors.

  3. Alignment-Free Methods for the Detection and Specificity Prediction of Adenylation Domains.

    PubMed

    Agüero-Chapin, Guillermin; Pérez-Machado, Gisselle; Sánchez-Rodríguez, Aminael; Santos, Miguel Machado; Antunes, Agostinho

    2016-01-01

    Identifying adenylation domains (A-domains) and their substrate specificity can aid the detection of nonribosomal peptide synthetases (NRPS) at genome/proteome level and allow inferring the structure of oligopeptides with relevant biological activities. However, that is challenging task due to the high sequence diversity of A-domains (~10-40 % of amino acid identity) and their selectivity for 50 different natural/unnatural amino acids. Altogether these characteristics make their detection and the prediction of their substrate specificity a real challenge when using traditional sequence alignment methods, e.g., BLAST searches. In this chapter we describe two workflows based on alignment-free methods intended for the identification and substrate specificity prediction of A-domains. To identify A-domains we introduce a graphical-numerical method, implemented in TI2BioP version 2.0 (topological indices to biopolymers), which in a first step uses protein four-color maps to represent A-domains. In a second step, simple topological indices (TIs), called spectral moments, are derived from the graphical representations of known A-domains (positive dataset) and of unrelated but well-characterized sequences (negative set). Spectral moments are then used as input predictors for statistical classification techniques to build alignment-free models. Finally, the resulting alignment-free models can be used to explore entire proteomes for unannotated A-domains. In addition, this graphical-numerical methodology works as a sequence-search method that can be ensemble with homology-based tools to deeply explore the A-domain signature and cope with the diversity of this class (Aguero-Chapin et al., PLoS One 8(7):e65926, 2013). The second workflow for the prediction of A-domain's substrate specificity is based on alignment-free models constructed by transductive support vector machines (TSVMs) that incorporate information of uncharacterized A-domains. The construction of the models was

  4. Adenylate cyclase orthologues in two filamentous entomopathogens contribute differentially to growth, conidiation, pathogenicity, and multistress responses.

    PubMed

    Wang, Jie; Zhou, Gang; Ying, Sheng-Hua; Feng, Ming-Guang

    2014-04-01

    Adenylate cyclase (AC) is a core element of cAMP signalling network. Here we show functional diversity and differentiation of Beauveria bassiana AC (BbAC) and Metarhizium robertsii AC (MrAC). Severe growth defects occurred in ΔBbAC and ΔMrAC grown on nutrition-rich SDAY and several minimal media but were largely alleviated by adding cAMP to SDAY. Conidial yield increased greatly in ΔBbAC but decreased in ΔMrAC. During colony growth, ΔBbAC was highly sensitive to oxidation, high osmolarity, cell wall perturbation, carbendazim fungicide, Mn(2+), Zn(2+), Fe(3+), and EDTA but more tolerant to Cu(2+) while ΔMrAC showed higher osmotolerance, decreased sensitivity to Fe(3+), and null response to carbendazim or cell wall stress despite similar responses to oxidation and other metal ions. Conidial UV-B resistance decreased by 32% in ΔBbAC and 22% in ΔMrAC despite little change in their theromotolerance. Median lethal time (LT50) estimates of ΔBbAC and ΔMrAC against susceptible insects were 10.9 and 1.4 d longer than those from wild-type strains respectively. All the phenotypic changes were restored to wild-type levels by each gene complementation. Taken together, BbAC and MrAC regulated differentially conidiation, pathogenicity, and multistress responses in B. bassiana and M. robertsii, thereby making different contributions to their biocontrol potential. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  5. Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis.

    PubMed

    Barbier, Mariette; Boehm, Dylan T; Sen-Kilic, Emel; Bonnin, Claire; Pinheiro, Theo; Hoffman, Casey; Gray, Mary; Hewlett, Erik; Damron, F Heath

    2017-01-01

    Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions. Copyright © 2016 American Society for Microbiology.

  6. Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis

    PubMed Central

    Barbier, Mariette; Boehm, Dylan T.; Sen-Kilic, Emel; Bonnin, Claire; Pinheiro, Theo; Hoffman, Casey; Gray, Mary; Hewlett, Erik

    2016-01-01

    ABSTRACT Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions. PMID:27849178

  7. Quantification of the adenylate cyclase toxin of Bordetella pertussis in vitro and during respiratory infection.

    PubMed

    Eby, Joshua C; Gray, Mary C; Warfel, Jason M; Paddock, Christopher D; Jones, Tara F; Day, Shandra R; Bowden, James; Poulter, Melinda D; Donato, Gina M; Merkel, Tod J; Hewlett, Erik L

    2013-05-01

    Whooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ≈ 10(8) CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ≈ 10(8) CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ≈ 60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.

  8. Quantification of the Adenylate Cyclase Toxin of Bordetella pertussis In Vitro and during Respiratory Infection

    PubMed Central

    Eby, Joshua C.; Gray, Mary C.; Warfel, Jason M.; Paddock, Christopher D.; Jones, Tara F.; Day, Shandra R.; Bowden, James; Poulter, Melinda D.; Donato, Gina M.; Merkel, Tod J.

    2013-01-01

    Whooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ∼108 CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ∼108 CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ∼60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis. PMID:23429530

  9. Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating

    PubMed Central

    Hurley, Matthew M.; Maunze, Brian; Block, Megan E.; Frenkel, Mogen M.; Reilly, Michael J.; Kim, Eugene; Chen, Yao; Li, Yan; Baker, David A.; Liu, Qing-Song; Choi, SuJean

    2016-01-01

    While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive. PMID:27597817

  10. Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

    PubMed Central

    Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Devoisselle, Jean-Marie; Ladant, Daniel; Chopineau, Joel

    2013-01-01

    Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study’s in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions. PMID:24297899

  11. Hemodynamic actions of systemically injected pituitary adenylate cyclase activating polypeptide-27 in the rat

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The aims of this study were (1) to characterize the hemodynamic mechanisms underlying the hypotensive effects of pituitary adenylate cyclase activating polypeptide-27 (PACAP-27 0.1-2.0 nmol/kg, i.v.) in pentobarbital-anesthetized rats, and (2) to determine the roles of the autonomic nervous system, adrenal catecholamines and endothelium-derived nitric oxide (NO) in the expression of PACAP-27-mediated effects on hemodynamic function. PACAP-27 produced dose-dependent decreases in mean arterial blood pressure and hindquarter and mesenteric vascular resistances in saline-treated rats. PACAP-27 also produced pronounced falls in mean arterial blood pressure in rats treated with the ganglion blocker, chlorisondamine (5 mg/kg, i.v.). The hypotensive and vasodilator actions of PACAP-27 were not attenuated by the beta-adrenoceptor antagonist, propranolol (1 mg/kg, i.v.), or the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME 50 micromol/kg, i.v.). PACAP-27 produced dose-dependent increases in heart rate whereas the hypotensive response produced by the nitrovasodilator, sodium nitroprusside (10 microg/kg, i.v.), was associated with a minimal tachycardia. The PACAP-27-induced tachycardia was unaffected by chlorisondamine, but was virtually abolished by propranolol. These results suggest that the vasodilator effects of PACAP-27 are due to actions in the microcirculation rather than to the release of adrenal catecholamines and that this vasodilation may not involve the release of endothelium-derived NO. These results also suggest that PACAP-27 produces tachycardia by directly releasing norepinephrine from cardiac sympathetic nerve terminals rather than by direct or baroreceptor reflex-mediated increases in sympathetic nerve activity.

  12. Modulation of a pre-existing conformational equilibrium tunes adenylate kinase activity.

    PubMed

    Ådén, Jörgen; Verma, Abhinav; Schug, Alexander; Wolf-Watz, Magnus

    2012-10-10

    Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AK(eco)) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turnover rate is inversely proportional to the lifetime of the active conformation. Therefore, structural plasticity is intimately coupled to enzymatic turnover in AK(eco). Here, we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate and, in turn, the turnover number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding subdomains results in a population shift toward the open conformation and a resulting increase of k(cat). Addition of the osmolyte TMAO to AK(eco) results in population shift toward the closed conformation and a significant reduction of k(cat). The Michaelis constants (K(M)) scale with the change in k(cat), which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, k(cat) and K(M) are mutually dependent, and in the case of AK(eco), any perturbation that modulates k(cat) is mirrored with a proportional response in K(M). Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affects enzymatic catalysis. From an evolutionary perspective, our findings suggest that, for AK(eco), there exists ample flexibility to obtain a specificity constant (k(cat)/K(M)) that commensurate with the exerted cellular selective pressure.

  13. Pituitary adenylate cyclase activating polypeptide: an important vascular regulator in human skin in vivo.

    PubMed

    Seeliger, Stephan; Buddenkotte, Jörg; Schmidt-Choudhury, Anjona; Rosignoli, Carine; Shpacovitch, Victoria; von Arnim, Ulrike; Metze, Dieter; Rukwied, Roman; Schmelz, Martin; Paus, Ralf; Voegel, Johannes J; Schmidt, Wolfgang E; Steinhoff, Martin

    2010-11-01

    Pituitary adenylate cyclase-activating peptide (PACAP) is an important neuropeptide and immunomodulator in various tissues. Although this peptide and its receptors (ie, VPAC1R, VPAC2R, and PAC1R) are expressed in human skin, their biological roles are unknown. Therefore, we tested whether PACAP regulates vascular responses in human skin in vivo. When injected intravenously, PACAP induced a significant, concentration-dependent vascular response (ie, flush, erythema, edema) and mediated a significant and concentration-dependent increase in intrarectal body temperature that peaked at 2.7°C. Topical application of PACAP induced marked concentration-dependent edema. Immunohistochemistry revealed a close association of PACAP-immunoreactive nerve fibers with mast cells and dermal blood vessels. VPAC1R was expressed by dermal endothelial cells, CD4+ and CD8+ T cells, mast cells, and keratinocytes, whereas VPAC2R was expressed only in keratinocytes. VPAC1R protein and mRNA were also detected in human dermal microvascular endothelial cells. The PACAP-induced change in cAMP production in these cells demonstrated VPAC1R to be functional. PACAP treatment of organ-cultured human skin strongly increased the number of CD31+ vessel cross-sections. Taken together, these results suggest that PACAP directly induces vascular responses that may be associated with neurogenic inflammation, indicating for the first time that PACAP may be a crucial vascular regulator in human skin in vivo. Antagonists to PACAP function may be beneficial for the treatment of inflammatory skin diseases with a neurogenic component.

  14. Pituitary adenylate cyclase-activating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation and glucohomeostasis.

    PubMed

    Hamelink, Carol; Tjurmina, Olga; Damadzic, Ruslan; Young, W Scott; Weihe, Eberhard; Lee, Hyeon-Woo; Eiden, Lee E

    2002-01-08

    The adrenal gland is important for homeostatic responses to metabolic stress: hypoglycemia stimulates the splanchnic nerve, epinephrine is released from adrenomedullary chromaffin cells, and compensatory glucogenesis ensues. Acetylcholine is the primary neurotransmitter mediating catecholamine secretion from the adrenal medulla. Accumulating evidence suggests that a secretin-related neuropeptide also may function as a transmitter at the adrenomedullary synapse. Costaining with highly specific antibodies against the secretin-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and the vesicular acetylcholine transporter (VAChT) revealed that PACAP is found in nerve terminals at all mouse adrenomedullary cholinergic synapses. Mice with a targeted deletion of the PACAP gene had otherwise normal cholinergic innervation and morphology of the adrenal medulla, normal adrenal catecholamine and blood glucose levels, and an intact initial catecholamine secretory response to insulin-induced hypoglycemia. However, insulin-induced hypoglycemia was more profound and longer-lasting in PACAP knock-outs, and was associated with a dose-related lethality absent in wild-type mice. Failure of PACAP-deficient mice to adequately counterregulate plasma glucose levels could be accounted for by impaired long-term secretion of epinephrine, secondary to a lack of induction of tyrosine hydroxylase, normally occurring after insulin hypoglycemia in wild-type mice, and a consequent depletion of adrenomedullary epinephrine stores. Thus, PACAP is needed to couple epinephrine biosynthesis to secretion during metabolic stress. PACAP appears to function as an "emergency response" cotransmitter in the sympathoadrenal axis, where the primary secretory response is controlled by a classical neurotransmitter but sustained under paraphysiological conditions by a neuropeptide.

  15. Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

    PubMed Central

    Schulze, Silvia; Fischer, André; Grundmann, Ursula; Alutis, Marie; Kühl, Anja A.; Tamas, Andrea; Toth, Gabor; Dunay, Miklos P.; Göbel, Ulf B.; Reglodi, Dora; Bereswill, Stefan

    2014-01-01

    Background The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis. Methodology/Principal Findings Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner. Conclusion/Significance Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases. PMID:25238233

  16. Biochemical mechanisms of myocardial adenylate cyclase subsensitivity to isoproterenol in cardiac hypertrophy of spontaneously hypertensive rats

    SciTech Connect

    Cheon, J.W.

    1986-01-01

    The responsiveness of the myocardial adenylate cyclase (AC) system in generating cAMP was studied using isoproterenol (a beta-adrenergic receptor agonist), cholera toxin (a guanosinetriphosphatase inhibitor) and forskolin (a catalytic unit activator) in isolated myocytes of age-matched, 14-17 weeks old Wistar Kyoto normotensive rates (WKYs) and spontaneously hypertensive rats (SHRs). We found a reduction in isoproterenol-stimulated cAMP formation in myocytes of SHRs compared with WKYs. This reduction was not due to changes in isoproterenol-receptor interactions. Scatchard plot analysis of (/sup 3/H)CGP 12177 binding to beta-adrenergic receptors in isolated myocytes of WKYs and SHRs revealed to significant differences in the maximum number of binding sites or dissociation constant. There were no significant differences in Ki and IC/sub 50/ calculated from the competitive displacement of (/sup 3/H)CGP 12177 binding by (-) isoproterenol, suggesting no change in the affinity of the beta-adrenergic receptors for isoproterenol. We found no significant differences in forskolin-stimulated cAMP formation between the two groups. This suggest that the reduction in isoproterenol-stimulated cAMP formation observed in myocytes of SHRs is not due to changes in the ability of catalytic unit to convert ATP to cAMP. Interestingly, cholera toxin-stimulated cAMP formation was increased in myocytes of SHRs. One possible explanation for these observations may be increased guanosinetriphosphatase (GTPase) activation by isoproterenol in myocytes of SHRs. The activation of GTPase by isoproterenol in myocytes of SHRs. The activation of GTPase by isoproterenol was measured as the release of Pi from (..gamma..-/sup 32/P)GTP. There was an increase in isoproterenol-stimulated GTPase activity in myocytes of SHRs compared with WKYs.

  17. Homologous desensitization of adenylate cyclase: the role of. beta. -adrenergic receptor phosphorylation and dephosphorylation

    SciTech Connect

    Sibley, D.R.; Strasser, R.H.; Daniel, K.; Lefkowitz, R.J.

    1986-03-05

    The authors utilized the frog erythrocyte (FE) as a ..beta..-adreneric receptor (..beta..AR) model system in which to study homologous desensitization. Preincubation with isoproterenol (ISO) leads to a 50% decline in ISO-stimulated adenylate cyclase (AC) activity without significant changes in basal, PGE/sub 1/-, NaF-, GppNHp-, forskolin-, or MnCl/sub 2/-stimulated AC activities. ISO treatment also induces the sequestration of ..beta..AR from the cell surface as evidenced by a 35% decline in (/sup 3/H)CGP-12177 binding sites on the surface of intact FE. Treatment of intact FE with ISO also promotes ..beta..AR phosphorylation to 2 mol PO/sub 4//mol of ..beta..AR. At 25/sup 0/C, the time courses of ISO-induced AC desensitization, ..beta..AR sequestration and ..beta..AR phosphorylation are identical occurring without a lag and exhibiting a t 1/2 of 30 min and a maximal response at 2.5 hrs. The sequestered ..beta..AR can be partially recovered upon cell lysis in a light membrane fraction (LMF), separable from the plasma membranes using sucrose gradients or differential centrifugation. ..beta..AR phosphorylation is reversed in the sequestered LMF exhibiting a PO/sub 4//..beta..AR stoichiometry of 0.7 mol/mol - similar to that observed under basal conditions. These data suggest that phosphorylation of ..beta..AR in the plasma membrane promotes their translocation away from the cell surface into a sequestered membrane domain where the phosphorylation is reversed, thus, enabling the return of ..beta..AR back to the cell surface and recoupling with AC.

  18. Rapid kinetics of 2-adrenergic agonist binding and inhibition of adenylate cyclase

    SciTech Connect

    Thomsen, W.; Neubig, R.R.

    1987-05-01

    Activation of 2-adrenergic receptors in human platelets results in inhibition of adenylate cyclase (AC). To elucidate the relation between agonist binding and response, the authors have used a novel rapid-mix quench method to compare the kinetics of binding and response. At functionally effective concentrations, the time course of binding of the full 2-agonist, (TH)UK14,304 (UK), to purified platelet membranes was faster than could be measured manually. Using the rapid-mix quench method, agonist binding was quantitated for times for 0.3 to 60 seconds. UK binding exhibited biexponential kinetics. The rate constant of the fast binding component increases linearly with agonist concentration from 1 to 100 nM with a second order rate constant and 7 x 10WM s (at 25C). The slow rate constant was nearly independent of agonist concentration. The half times of the fast and slow components of binding for 100 nM UK are 1.5 seconds and approximately 2 minutes respectively. The rate and magnitude of the fast binding was unaffected by 10 M GTP whereas the magnitude of the slow phase was markedly reduced. Inhibition of forskolin stimulated AC by 100 M epinephrine occurs with a lag of 5-10 seconds in the presence of 10 M GTP. At lower GTP concentrations, this lag is prolonged. The observation that the fast component of agonist binding precedes inhibition even at agonist concentrations 20-fold lower than the EC40 for responses indicates that the rate limiting step in inhibition of AC is distal to the binding of agonist.

  19. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice.

    PubMed

    Hattori, Satoko; Takao, Keizo; Tanda, Koichi; Toyama, Keiko; Shintani, Norihito; Baba, Akemichi; Hashimoto, Hitoshi; Miyakawa, Tsuyoshi

    2012-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC(1)). Recent studies reveal that genetic variants of the PACAP and PAC(1) genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory.

  20. Molecular cloning and expression of a chicken pituitary adenylate cyclase-activating polypeptide receptor.

    PubMed

    Peeters, K; Gerets, H H; Princen, K; Vandesande, F

    1999-08-25

    Although, since the isolation of pituitary adenylate cyclase-activating polypeptide (PACAP), a wealth of literature has been published describing its localization, binding sites, and biological activities in a variety of mammalian tissues, only very little is known about PACAP in avian species. Therefore, in order to find out the sites of actions of PACAP and to elucidate its physiological significance in birds, we identified a chicken PACAP receptor homologue of the mammalian type I receptors (PAC(1)-Rs). The chicken PACAP type I cDNA sequence was obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with 3'- and 5'-RACE PCR. This cDNA encodes a 471 amino acid precursor protein, sharing 81-83% sequence identity with mammalian analogs and 76% amino acid identity with the goldfish type I PACAP receptor. Northern blot analysis of chicken brain poly(A)(+)-rich RNA revealed the presence of a 5.5 kb and 7.5 kb PAC(1) receptor transcript. RT-PCR revealed that the chicken PACAP receptor is mainly expressed in the brain and gonads. A smaller amount of the receptor mRNA was found in pituitary, adrenal gland, kidney, intestine, pancreas, lung, and heart tissue. In situ hybridization with specific antisense oligodeoxynucleotide probes showed a widespread distribution of PAC(1) receptor mRNA in the chicken brain, with the highest expression being found in the dorsal telencephalon, olfactory bulb, hypothalamus, optic tectum, and cerebellar cortex. These findings suggest that PACAP affect a variety of functions both in the brain and peripheral tissues of the chicken.

  1. Characteristics in limbic encephalitis with anti-adenylate kinase 5 autoantibodies.

    PubMed

    Do, Le-Duy; Chanson, Eve; Desestret, Virginie; Joubert, Bastien; Ducray, François; Brugière, Sabine; Couté, Yohann; Formaglio, Maité; Rogemond, Veronique; Thomas-Antérion, Catherine; Borrega, Laura; Laurens, Brice; Tison, Francois; Curot, Jonathan; De Brouker, Thomas; Lebrun-Frenay, Christine; Delattre, Jean-Yves; Antoine, Jean-Christophe; Honnorat, Jerome

    2017-02-07

    To report 10 patients with limbic encephalitis (LE) and adenylate kinase 5 autoantibodies (AK5-Abs). We conducted a retrospective study in a cohort of 50 patients with LE with uncharacterized autoantibodies and identified a specific target using immunohistochemistry, Western blotting, immunoprecipitation, mass spectrometry, and cell-based assay. AK5 (a known autoantigen of LE) was identified as the target of antibodies in the CSFs and sera of 10 patients with LE (median age 64 years; range 57-80), which was characterized by subacute anterograde amnesia without seizure and sometimes preceded by a prodromal phase of asthenia or mood disturbances. Anterograde amnesia can be isolated, but some patients also complained of prosopagnosia, paroxysmal anxiety, or abnormal behavior. No associated cancer was observed. All 10 patients had bilateral hippocampal hypersignal on a brain MRI. CSF analysis generally showed a mild pleiocytosis with elevated immunoglobulin G index and oligoclonal bands, as well as high levels of tau protein with normal concentration of Aβ42 and phospho-tau, suggesting a process of neuronal death. Except for one patient, clinical response to immunotherapy was unfavorable, with persistence of severe anterograde amnesia. Two patients evolved to severe cognitive decline. Hippocampal atrophy was observed on control brain MRI. Using in vitro tests on hippocampal neurons, we did not identify clues suggesting a direct pathogenic role of AK5-Abs. AK5-Abs should be systematically considered in aged patients with subacute anterograde amnesia. Recognition of this disorder is important to develop new treatment strategies to prevent irreversible limbic damage. © 2017 American Academy of Neurology.

  2. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice

    PubMed Central

    Hattori, Satoko; Takao, Keizo; Tanda, Koichi; Toyama, Keiko; Shintani, Norihito; Baba, Akemichi; Hashimoto, Hitoshi; Miyakawa, Tsuyoshi

    2012-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1). Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory. PMID:23060763

  3. Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family.

    PubMed

    Martín, César; Requero, M-Asunción; Masin, Jiri; Konopasek, Ivo; Goñi, Félix M; Sebo, Peter; Ostolaza, Helena

    2004-06-01

    Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers.

  4. Hemodynamic actions of systemically injected pituitary adenylate cyclase activating polypeptide-27 in the rat

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The aims of this study were (1) to characterize the hemodynamic mechanisms underlying the hypotensive effects of pituitary adenylate cyclase activating polypeptide-27 (PACAP-27 0.1-2.0 nmol/kg, i.v.) in pentobarbital-anesthetized rats, and (2) to determine the roles of the autonomic nervous system, adrenal catecholamines and endothelium-derived nitric oxide (NO) in the expression of PACAP-27-mediated effects on hemodynamic function. PACAP-27 produced dose-dependent decreases in mean arterial blood pressure and hindquarter and mesenteric vascular resistances in saline-treated rats. PACAP-27 also produced pronounced falls in mean arterial blood pressure in rats treated with the ganglion blocker, chlorisondamine (5 mg/kg, i.v.). The hypotensive and vasodilator actions of PACAP-27 were not attenuated by the beta-adrenoceptor antagonist, propranolol (1 mg/kg, i.v.), or the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME 50 micromol/kg, i.v.). PACAP-27 produced dose-dependent increases in heart rate whereas the hypotensive response produced by the nitrovasodilator, sodium nitroprusside (10 microg/kg, i.v.), was associated with a minimal tachycardia. The PACAP-27-induced tachycardia was unaffected by chlorisondamine, but was virtually abolished by propranolol. These results suggest that the vasodilator effects of PACAP-27 are due to actions in the microcirculation rather than to the release of adrenal catecholamines and that this vasodilation may not involve the release of endothelium-derived NO. These results also suggest that PACAP-27 produces tachycardia by directly releasing norepinephrine from cardiac sympathetic nerve terminals rather than by direct or baroreceptor reflex-mediated increases in sympathetic nerve activity.

  5. Neurally released pituitary adenylate cyclase-activating polypeptide enhances guinea pig intrinsic cardiac neurone excitability.

    PubMed

    Tompkins, John D; Ardell, Jeffrey L; Hoover, Donald B; Parsons, Rodney L

    2007-07-01

    Intracellular recordings were made in vitro from guinea-pig cardiac ganglia to determine whether endogenous neuropeptides such as pituitary adenylate cyclase-activating polypeptide (PACAP) or substance P released during tetanic neural stimulation modulate cardiac neurone excitability and/or contribute to slow excitatory postsynaptic potentials (sEPSPs). When nicotinic and muscarinic receptors were blocked by hexamethonium and atropine, 20 Hz stimulation for 10 s initiated a sEPSP in all innervated neurones. In 40% of the cells, excitability was enhanced after termination of the sEPSP. This suggested that non-cholinergic receptor-mediated mechanisms contributed to the sEPSP and modulated neuronal excitability. Exogenous PACAP and substance P initiated a slow depolarization in the neurones whereas neuronal excitability was only increased by PACAP. When ganglia were treated with the PAC1 antagonist PACAP6-38 (500 nM), the sEPSP evoked by 20 Hz stimulation was reduced by approximately 50% and an enhanced excitability occurred in only 10% of the cells. These observations suggested that PACAP released from preganglionic nerve terminals during tetanic stimulation enhanced neuronal excitability and evoked sEPSPs. After addition of 1 nM PACAP to the bath, 7 of 9 neurones exhibited a tonic firing pattern whereas in untreated preparations, the neurons had a phasic firing pattern. PACAP6-38 (500 nM) diminished the increase in excitability caused by 1 nM PACAP so that only 4 of 13 neurones exhibited a tonic firing pattern and the other 9 cells retained a phasic firing pattern. These findings indicate that PACAP can be released by tetanic neural stimulation in vitro and increase the excitability of intrinsic cardiac neurones. We hypothesize that in vivo PACAP released during preganglionic firing may modulate neurotransmission within the intrinsic cardiac ganglia.

  6. Pituitary Adenylate Cyclase-Activating Polypeptide induces a depressive-like phenotype in rats

    PubMed Central

    Seiglie, Mariel P.; Smith, Karen L.; Blasio, Angelo; Cottone, Pietro; Sabino, Valentina

    2015-01-01

    Major Depressive Disorder (MDD) is a chronic, life-threatening psychiatric condition characterized by depressed mood, psychomotor alterations, and a markedly diminished interest or pleasure in most activities, known as anhedonia. Available pharmacotherapies have limited success and the need for new strategies is clear. Recent studies attribute a major role to the pituitary adenylate cyclase-activating polypeptide (PACAP) system in mediating the response to stress. PACAP knockout mice display profound alterations in depressive-like behaviors and genetic association studies have demonstrated that genetic variants of the PACAP gene are associated with MDD. However, the effects of PACAP on depressive-like behaviors in rodents have not yet been systematically examined. The present study investigated the effects of central administration of PACAP in rats on depressive-like behaviors, using well-established animal models that represent some of the endophenotypes of depression. We used intracranial self-stimulation (ICSS) to assess the brain reward function, saccharin preference test to assess anhedonia, social interaction to assess social withdrawal, and forced swim test (FST) to assess behavioral despair. PACAP raised the current threshold for ICSS, elevation blocked by the PACAP antagonist PACAP(6-38). PACAP reduced the preference for a sweet saccharin solution, and reduced the time the rats spent interacting with a novel animal. Interestingly, PACAP administration did not affect immobility in the FST. Our results demonstrate a role for the central PACAP/PAC1R system in the regulation of depressive-like behaviors, and suggest that hyperactivity of the PACAP/PAC1R system may contribute to the pathophysiology of depression, particularly the associated anhedonic symptomatology and social dysfunction. PMID:26264905

  7. Effects of UVB irradiation on epidermal adenylate cyclase responses in vitro: its relation to sunburn cell formation.

    PubMed

    Iizuka, H; Ishida-Yamamoto, A; Kajita, S; Tsutsui, M; Ohkuma, N

    1988-01-01

    UVB irradiation augmented the beta-adrenergic adenylate cyclase response of pig skin epidermis in vitro. The effect was observed 2-4 h following the irradiation and lasted at least for 48 h. There was no significant difference in cyclic AMP phosphodiesterase activity between control and UVB-irradiated epidermis at lower irradiation dose (150 mJ/cm2), which is the dose of the most marked beta-adrenergic augmentation effect. The augmentation effect was specific to the beta-adrenergic system; adenosine and histamine adenylate cyclase responses were unchanged or decreased depending on the irradiation dose. Histologically, marked sunburn-cell formation was observed following the UVB irradiation. It has been suggested that oxygen intermediates generated by ultraviolet radiation participate in sunburn-cell formation. The addition of superoxide dismutase (SOD) in the incubation medium significantly inhibited sunburn-cell formation. On the other hand, the beta-adrenergic augmentation effect was not affected by the addition of SOD. Other scavengers of oxygen intermediates (catalase, catalase + SOD, xanthine, or mannitol) did not inhibit the UVB-induced beta-adrenergic augmentation effect. Further, superoxide-anion generating systems (hypoxanthine-xanthine oxidase system and acetaldehyde-xanthine oxidase system) revealed no stimulatory effect on the beta-adrenergic response of epidermis. These results indicate that (a) the UVB-induced beta-adrenergic augmentation effect is inherent to skin and does not depend on systemic factors such as inflammatory infiltrates following UVB irradiation; (b) in contrast to sunburn-cell formation, induction of the beta-adrenergic adenylate cyclase response is not directly associated with oxygen intermediates generated by UVB irradiation.

  8. Adenylate cyclase A acting on PKA mediates induction of stalk formation by cyclic diguanylate at the Dictyostelium organizer

    PubMed Central

    Chen, Zhi-Hui; Singh, Reema; Cole, Christian; Lawal, Hajara Mohammed; Schilde, Christina; Febrer, Melanie; Barton, Geoffrey J.; Schaap, Pauline

    2017-01-01

    Coordination of cell movement with cell differentiation is a major feat of embryonic development. The Dictyostelium stalk always forms at the organizing tip, by a mechanism that is not understood. We previously reported that cyclic diguanylate (c-di-GMP), synthesized by diguanylate cyclase A (DgcA), induces stalk formation. Here we used transcriptional profiling of dgca− structures to identify target genes for c-di-GMP, and used these genes to investigate the c-di-GMP signal transduction pathway. We found that knockdown of cAMP-dependent protein kinase (PKA) activity in prestalk cells reduced stalk gene induction by c-di-GMP, whereas PKA activation bypassed the c-di-GMP requirement for stalk gene expression. c-di-GMP caused a persistent increase in cAMP, which still occurred in mutants lacking the adenylate cyclases ACG or ACR, or the cAMP phosphodiesterase RegA. However, both inhibition of adenylate cyclase A (ACA) with SQ22536 and incubation of a temperature-sensitive ACA mutant at the restrictive temperature prevented c-di-GMP–induced cAMP synthesis as well as c-di-GMP–induced stalk gene transcription. ACA produces the cAMP pulses that coordinate Dictyostelium morphogenetic cell movement and is highly expressed at the organizing tip. The stalk-less dgca− mutant regained its stalk by expression of a light-activated adenylate cyclase from the ACA promoter and exposure to light, indicating that cAMP is also the intermediate for c-di-GMP in vivo. Our data show that the more widely expressed DgcA activates tip-expressed ACA, which then acts on PKA to induce stalk genes. These results explain why stalk formation in Dictyostelia always initiates at the site of the morphogenetic organizer. PMID:28057864

  9. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    SciTech Connect

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh; Finley, Natosha L.

    2014-10-10

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex. Taken

  10. Acyclic phosphonate nucleotides and human adenylate kinases: impact of a borano group on alpha-P position.

    PubMed

    Topalis, D; Alvarez, K; Barral, K; Munier-Lehmann, H; Schneider, B; Véron, M; Guerreiro, C; Mulard, L; El-Amri, C; Canard, B; Deville-Bonne, D

    2008-04-01

    Adenylate kinases are involved in the activation of antiviral drugs such as the acyclic phosphonates analogs PMEA and (R)PMPA. We examine the in vitro phosphorylation of PMEA and PMPA bearing a borano- or a H- group on the phosphorus atom. The alpha-borano or alpha-H on PMEA and PMPA were detrimental to the activity of recombinant human AMP kinases 1 and 2. Docking PMEA to the active site of AMP kinase 1 indicated that the borano group may prevent two conserved critical Arg interactions with the alpha-phosphate, resulting in substrate bad positioning.

  11. Effect of cardiopulmonary bypass on beta adrenergic receptor-adenylate cyclase system on surfaces of peripheral lymphocytes.

    PubMed

    Luo, A; Tian, Y; Jin, S

    2000-01-01

    The experimental results showed that the level of CAMP, the ratio of cAPM to cGMP, IL-2R expression and IL-2 production in vitro in lymphocytes immediate and 2 weeks after cardiopulmonary bypass (CPB) were significantly lower than those before anesthetics in the patients undergoing cardiac surgery with CPB. These findings suggested that CPB could cause serious damage to adrenergic beta receptor-adenylate cyclase system on circulating lymphocytes surfaces, which might be one of the mechanisms resulting in immunosuppression after open heart surgery with CPB.

  12. Primary enzyme quantitation

    DOEpatents

    Saunders, G.C.

    1982-03-04

    The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

  13. Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis.

    PubMed

    Skopova, Karolina; Tomalova, Barbora; Kanchev, Ivan; Rossmann, Pavel; Svedova, Martina; Adkins, Irena; Bibova, Ilona; Tomala, Jakub; Masin, Jiri; Guiso, Nicole; Osicka, Radim; Sedlacek, Radislav; Kovar, Marek; Sebo, Peter

    2017-06-01

    The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b(+)) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b(-) cells. The nonhemolytic AC(+) Hly(-) bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC(+) Hly(-) mutant also infected mouse lungs as efficiently as the parental AC(+) Hly(+) strain. Hence, elevation of cAMP in CD11b(-) cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>10(7) CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent. Copyright © 2017 American Society for Microbiology.

  14. Inhibition of Siderophore Biosynthesis in Mycobacterium tuberculosis with Nucleoside Bisubstrate Analogues: Structure–Activity Relationships of the Nucleobase Domain of 5′-O-[N-(Salicyl)sulfamoyl]adenosine

    PubMed Central

    Neres, João; Labello, Nicholas P.; Somu, Ravindranadh V.; Boshoff, Helena I.; Wilson, Daniel J.; Vannada, Jagadeshwar; Chen, Liqiang; Barry, Clifton E.; Bennett, Eric M.; Aldrich, Courtney C.

    2009-01-01

    5′-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) is a prototype for a new class of antitubercular agents that inhibit the aryl acid adenylating enzyme (AAAE) known as MbtA involved in biosynthesis of the mycobactins. Herein, we report the structure-based design, synthesis, biochemical, and biological evaluation of a comprehensive and systematic series of analogues, exploring the structure–activity relationship of the purine nucleobase domain of Sal-AMS. Significantly, 2-phenyl-Sal-AMS derivative 26 exhibited exceptionally potent antitubercular activity with an MIC99 under iron-deficient conditions of 0.049 µM while the N-6-cyclopropyl-Sal-AMS 16 led to improved potency and to a 64-enhancement in activity under iron-deficient conditions relative to iron-replete conditions, a phenotype concordant with the designed mechanism of action. The most potent MbtA inhibitors disclosed here display in vitro antitubercular activity superior to most current first line TB drugs, and these compounds are also expected to be useful against a wide range of pathogens that require aryl-capped siderphores for virulence. PMID:18690677

  15. Red cell enzyme polymorphisms in Papua New Guinea Eastern Highlands.

    PubMed

    Seger, J; Godelier, M; Halle, L; Lemonnier, P; Lory, J L; Rouger, P; Ruffie, J; Salmon, D

    1988-01-01

    Ten red cell enzyme polymorphisms, malic dehydrogenase (MDH1), adenylate kinase (AK), phosphohexose isomerase (PHI), adenosine deaminase (ADA), esterase D (ESD), glutamic pyruvic transaminase (GPT), acid phosphatase (ACP1), phosphoglucomutase 1 and 2 (PGM1, PGM2), phosphogluconate dehydrogenase (PGD) were investigated in the Baruya tribe and several Anga tribes living high in the Wonenara and Marawaka valleys in Papua New Guinea Eastern Highlands (6.5S, 145.5E). Also a non-Anga tribe, the Aziana or Kenaze, was sampled. Variants were observed in ADA, PGM1 and PGM2. AK and PHI were monomorphic, all subjects being AK 1 and PHI 1; MDH1 was also monomorphic in Anga while variants were observed in Aziana. This latter tribe differed markedly in each system from the Anga peoples.

  16. Adrenalectomy mediated alterations in adrenergic activation of adenylate cyclase in rat liver

    SciTech Connect

    El-Refai, M.; Chan, T.

    1986-05-01

    Adrenalectomy caused a large increase in the number of ..beta..-adrenergic binding sites on liver plasma membranes as measured by /sup 125/I-iodocyanopindolol (22 and 102 fmol/mg protein for control and adrenalectomized (ADX) rats). Concomitantly an increase in the number of binding sites for /sup 3/H-yohimbine was also observed (104 and 175 fmol/mg protein for control and adx membranes). Epinephrine-stimulated increase in cyclic AMP accumulation in isolated hepatocytes were greater in cells from ADX rats. This increase in ..beta..-adrenergic mediated action was much less than what may be expected as a result of the increase in the ..beta..-adrenergic binding in ADX membranes. In addition phenoxybenzamine (10 ..mu..M) further augmented this action of epinephrine in both control and ADX cells. To test the hypothesis that the increase in the number of the inhibitory ..cap alpha../sub 2/-adrenergic receptors in adrenalectomy is responsible for the muted ..beta..-adrenergic response, the authors injected rats with pertussis toxin (PT). This treatment may cause the in vivo ribosylation of the inhibitory binding protein (Ni). Adenylate cyclase (AC) activity in liver plasma membranes prepared from treated and untreated animals was measured. In contrast with control rats, treatment of ADX rats with PT resulted in a significant increase in the basal activity of AC (5.5 and 7.7 pmol/mg protein/min for untreated and treated rats respectively). Isoproterenol (10 ..mu..M), caused AC activity to increase to 6.5 and 8.4 pmol/mg protein/min for membranes obtained from ADX untreated and ADX treated rats respectively. The ..cap alpha..-adrenergic antagonists had no significant effect on the ..beta..-adrenergic-mediated activation of AC in liver plasma membranes from PT treated control and ADX rats. The authors conclude that the ..beta..-adrenergic activation of AC is attenuated by Ni protein both directly and as a result of activation of ..cap alpha..-adrenergic receptors.

  17. Part II: Biochemical changes after pituitary adenylate cyclase-activating polypeptide-38 infusion in migraine patients.

    PubMed

    Guo, Song; Vollesen, Anne Luise Haulund; Hansen, Young Bae Lee; Frandsen, Erik; Andersen, Malene Rohr; Amin, Faisal Mohammad; Fahrenkrug, Jan; Olesen, Jes; Ashina, Messoud

    2017-02-01

    Background Intravenous infusion of pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) provokes migraine attacks in 65-70% of migraine without aura (MO) patients. We investigated whether PACAP38 infusion causes changes in the endogenous production of PACAP38, vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), tumour necrosis factor alpha (TNFα), S100 calcium binding protein B (S100B), neuron-specific enolase and pituitary hormones in migraine patients. Methods We allocated 32 previously genotyped MO patients to receive intravenous infusion PACAP38 (10 pmol/kg/minute) for 20 minutes and recorded migraine-like attacks. Sixteen of the patients were carriers of the risk allele rs2274316 ( MEF2D), which confers increased risk of MO and may regulate PACAP38 expression, and 16 were non-carriers. We collected blood samples at baseline and 20, 30, 40, 60 and 90 minutes after the start of the infusion. A control group of six healthy volunteers received intravenous saline. Results PACAP38 infusion caused significant changes in plasma concentrations of VIP ( p = 0.026), prolactin ( p = 0.011), S100B ( p < 0.001) and thyroid-stimulating hormone (TSH; p = 0.015), but not CGRP ( p = 0.642) and TNFα ( p = 0.535). We found no difference in measured biochemical variables after PACAP38 infusion in patients who later developed migraine-like attacks compared to those who did not ( p > 0.05). There was no difference in the changes of biochemical variables between patients with and without the MEF2D-associated gene variant ( p > 0.05). Conclusion PACAP38 infusion elevated the plasma levels of VIP, prolactin, S100B and TSH, but not CGRP and TNFα. Development of delayed migraine-like attacks or the presence of the MEF2D gene variant was not associated with pre-ictal changes in plasma levels of neuropeptides, TNFα and pituitary hormones.

  18. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K.; Zhang, Yang; Banerjee, A.; Pinchuk, Grigoriy; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, S. oneidensis MR-1 uses the cAMP receptor protein, CRP, for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an E. coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, DMSO, or Fe(III), whereas the deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III), and to a lesser extent with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and the cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagella biosynthesis, and electron transport, were differentially expressed in the cyaC mutant, but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration, and may contribute to additional signaling pathways independent of CRP.

  19. Involvement of a membrane-bound class III adenylate cyclase in regulation of anaerobic respiration in Shewanella oneidensis MR-1.

    PubMed

    Charania, M A; Brockman, K L; Zhang, Y; Banerjee, A; Pinchuk, G E; Fredrickson, J K; Beliaev, A S; Saffarini, D A

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

  20. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K. L.; Zhang, Y.; Banerjee, A.; Pinchuk, Grigoriy E.; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

  1. Inhibition of adenylate cyclase by delta 9-tetrahydrocannabinol in mouse spleen cells: a potential mechanism for cannabinoid-mediated immunosuppression.

    PubMed

    Schatz, A R; Kessler, F K; Kaminski, N E

    1992-01-01

    The ability of delta 9-Tetrahydrocannabinol (delta 9-THC) to modulate adenylate cyclase activity in mouse spleen cells was investigated. These studies were prompted by the recent identification and cloning of a G-protein coupled cannabinoid receptor localized in certain regions of the brain and the potential for a common mechanism between cannabinoid-mediated CNS effects and immunosuppression. Temporal addition studies were initially performed to identify the period of time when spleen cells in culture were most susceptible to the inhibitory effects of delta 9-THC, as measured by the day 5 IgM antibody forming cell response. delta 9-THC was only inhibitory when added to spleen cell cultures during the first 2 hr following antigen sensitization. In light of this time course, adenylate cyclase activity was measured in spleen cells incubated in the presence of 22 microM delta 9-THC for 5 min and subsequently stimulated with forskolin. delta 9-THC treated spleen cells demonstrated a 33% inhibition and a 66% inhibition in intracellular cAMP after a 5 or 15 min stimulation with forskolin, respectively. These studies suggest that inhibition of immune function by delta 9-THC may be mediated through the inhibition of intracellular cAMP early after antigen stimulation.

  2. Vibrio vulnificus Biotype 3 Multifunctional Autoprocessing RTX Toxin Is an Adenylate Cyclase Toxin Essential for Virulence in Mice

    PubMed Central

    Ziolo, Kevin J.; Jeong, Hee-Gon; Kwak, Jayme S.; Yang, Shuangni; Lavker, Robert M.

    2014-01-01

    Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase. PMID:24614656

  3. Vibrio vulnificus biotype 3 multifunctional autoprocessing RTX toxin is an adenylate cyclase toxin essential for virulence in mice.

    PubMed

    Ziolo, Kevin J; Jeong, Hee-Gon; Kwak, Jayme S; Yang, Shuangni; Lavker, Robert M; Satchell, Karla J F

    2014-05-01

    Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase.

  4. Evidence for a presynaptic adenylate cyclase system facilitating (TH)norepinephrine release from rat brain neocortex slices and synaptosomes

    SciTech Connect

    Schoffelmeer, A.N.; Hogenboom, F.; Mulder, A.H.

    1985-10-01

    The effects of drugs known to enhance intracellular cyclic AMP levels on depolarization-induced (TH)norepinephrine release from superfused rat neocortical slices and synaptosomes were investigated. The adenylate cyclase activator forskolin, the membrane-permeating cyclic AMP analogues 8-bromo-cyclic AMP and dibutyryl cyclic AMP, as well as the phosphodiesterase inhibitors isobutylmethylxanthine and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrolidone (ZK 62771) enhanced the electrically evoked release of (TH)norepinephrine from superfused rat brain neocortex slices. 8-Bromo-cyclic GMP was without effect on the electrically evoked release. When (TH)norepinephrine release was enhanced by prolonging the electrical pulse duration from 2 msec to 10 msec, the relative inhibitory effect of the CaS channel blocker CdS and the relative facilitatory effect of the K+ channel blocker 4-aminopyridine remained unaffected. In striking contrast, the relative facilitatory effects of forskolin and 8-bromo-cyclic AMP were strongly reduced, whereas the effect of ZK 62771 was almost doubled. When veratrine-induced release of (TH)norepinephrine from cortex synaptosomes was examined, the facilitatory effects of forskolin, 8-bromo-cyclic AMP, and ZK 62771 were even more pronounced than in brain slices. The data strongly support the hypothesis that a presynaptic adenylate cyclase system plays a facilitatory role in the stimulus-secretion coupling process in central noradrenergic nerve terminals.

  5. Vasoactive intestinal polypeptide requires parallel changes in adenylate cyclase and phospholipase C to entrain circadian rhythms to a predictable phase

    PubMed Central

    An, Sungwon; Irwin, Robert P.; Allen, Charles N.; Tsai, Connie

    2011-01-01

    Circadian oscillations in the suprachiasmatic nucleus (SCN) depend on transcriptional repression by Period (PER)1 and PER2 proteins within single cells and on vasoactive intestinal polypeptide (VIP) signaling between cells. Because VIP is released by SCN neurons in a circadian pattern, and, after photic stimulation, it has been suggested to play a role in the synchronization to environmental light cycles. It is not known, however, if or how VIP entrains circadian gene expression or behavior. Here, we tested candidate signaling pathways required for VIP-mediated entrainment of SCN rhythms. We found that single applications of VIP reset PER2 rhythms in a time- and dose-dependent manner that differed from light. Unlike VIP-mediated signaling in other cell types, simultaneous antagonism of adenylate cyclase and phospholipase C activities was required to block the VIP-induced phase shifts of SCN rhythms. Consistent with this, VIP rapidly increased intracellular cAMP in most SCN neurons. Critically, daily VIP treatment entrained PER2 rhythms to a predicted phase angle within several days, depending on the concentration of VIP and the interval between VIP applications. We conclude that VIP entrains circadian timing among SCN neurons through rapid and parallel changes in adenylate cyclase and phospholipase C activities. PMID:21389307

  6. Dual actions of (-)-stepholidine on the dopamine receptor-mediated adenylate cyclase activity in rat corpus striatum.

    PubMed

    Dong, Z J; Guo, X; Chen, L J; Han, Y F; Jin, G Z

    1997-01-01

    (-)-Stepholidine (SPD) is an antagonist of normosensitive dopamine (DA) receptors, but it exhibits D1 agonistic action on rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta (SNC). In the present study, agonistic and antagonistic effects of SPD on the DA receptor-mediated synaptosomal adenylate cyclase (AC) activity in rat striatum were investigated. After blockade of D2 receptors, SPD augmented AC activity dose-dependently. The EC50 value was 41.1 +/- 8.6 micromol/L. At the concentration of 10 micromol/L, SPD increased cAMP formation from a basal level (50.8 +/- 10.3 pmol/mg protein/min) to 133.7 +/- 31.8 pmol/mg protein/min. The SPD-induced stimulation of AC activity was almost completely reversed by 10 micromol/L Sch23390. These results indicate that SPD possesses an agonistic action on the D1 receptor. Forskolin-stimulated adenylate cyclase (FSAC) activity was used as a model to elucidate the effect of SPD on D2 receptors. The results indicate that DA inhibited FSAC activity dose-dependently, while SPD partially restored FSAC activity. Taken together, these results support the conclusion that SPD has dual actions on DA receptors that mediate AC activity, i.e., an agonistic action on D1 receptors and an antagonistic action on D2 receptors.

  7. Structural basis for catalytically restrictive dynamics of a high-energy enzyme state

    PubMed Central

    Kovermann, Michael; Ådén, Jörgen; Grundström, Christin; Elisabeth Sauer-Eriksson, A.; Sauer, Uwe H.; Wolf-Watz, Magnus

    2015-01-01

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions. PMID:26138143

  8. Structural basis for catalytically restrictive dynamics of a high-energy enzyme state.

    PubMed

    Kovermann, Michael; Ådén, Jörgen; Grundström, Christin; Sauer-Eriksson, A Elisabeth; Sauer, Uwe H; Wolf-Watz, Magnus

    2015-07-03

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or 'invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power--a key aspect in rational design of enzymes catalysing novel reactions.

  9. Alternating Hemiplegia of Childhood as a New Presentation of Adenylate Cyclase 5-Mutation-Associated Disease: A Report of Two Cases.

    PubMed

    Westenberger, Ana; Max, Christoph; Brüggemann, Norbert; Domingo, Aloysius; Grütz, Karen; Pawlack, Heike; Weissbach, Anne; Kühn, Andrea A; Spiegler, Juliane; Lang, Anthony E; Sperner, Jürgen; Fung, Victor S C; Schallner, Jens; Gillessen-Kaesbach, Gabriele; Münchau, Alexander; Klein, Christine

    2017-02-01

    Mutations in the adenylate cyclase 5 (ADCY5) gene recently have been identified as the cause of a childhood-onset disorder characterized by persistent or paroxysmal choreic, myoclonic, and/or dystonic movements. The 2 novel mutations we identified expand the clinical spectrum of ADCY5 mutations to include alternating hemiplegia of childhood. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Laboratory Evaluation of Adenylate Energy Charge as a Test for Stress in Mytilus edulis and Nephtys incisa Treated with Dredged Material.

    DTIC Science & Technology

    1985-02-01

    concentrations of three adenine nucleotides, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP), which are...that all trace metals but iron were eliminated and the concentration of the vitamins thiamin and B12 were doubled. Adenylate Extraction 13. The adductor

  11. Adenylate cyclase, cyclic AMP and extracellular-signal-regulated kinase-2 in airway smooth muscle: modulation by protein kinase C and growth serum.

    PubMed Central

    Moughal, N; Stevens, P A; Kong, D; Pyne, S; Pyne, N J

    1995-01-01

    Bradykinin and phorbol 12-myristate 13-acetate stimulate adenylate cyclase activity in serum-depleted cultured airway smooth muscle via a protein kinase C (PKC)-dependent pathway. The probable target is the type II adenylate cyclase, which can integrate coincident signals from both PKC and Gs. Therefore, activation of Gs (by cholera-toxin pre-treatment) amplified the bradykinin-stimulated cyclic AMP signal and concurrently attenuated the partial activation of extracellular-signal-regulated kinase-2 (ERK-2) by bradykinin. We have previously demonstrated that, in order to induce full activation of ERK-2 with bradykinin, it is necessary to obliterate PKC-stimulated cyclic AMP formation. We concluded that the cyclic AMP signal limits the magnitude of ERK-2 activation [Pyne, Moughal, Stevens, Tolan and Pyne (1994) Biochem. J. 304, 611-616]. The present study indicates that the bradykinin-stimulated ERK-2 pathway is entirely cyclic AMP-sensitive, and suggests that coincident signal detection by adenylate cyclase may be an important physiological route for the modulation of early mitogenic signalling. Furthermore, the direct inhibition of adenylate cyclase activity enables bradykinin to induce DNA synthesis, indicating that the PKC-dependent activation of adenylate cyclase limits entry of cells into the cell cycle. These studies suggest that the mitogenicity of an agonist may be governed, in part, by its ability to stimulate an inhibitory cyclic AMP signal pathway in the cell. The activation of adenylate cyclase by PKC appears to be downstream of phospholipase D. However, in cells that were maintained in growth serum (i.e. were not growth-arrested), bradykinin was unable to elicit a PKC-stimulated cyclic AMP response. The lesion in the signal-response coupling was not at the level of either the receptor or phospholipase D, which remain functionally operative and suggests modification occurs at either PKC or adenylate cyclase itself. These studies are discussed with

  12. AMP promotes oxygen consumption and ATP synthesis in heart mitochondria through the adenylate kinase reaction: an NMR spectroscopy and polarography study.

    PubMed

    Doliba, Nicolai M; Babsky, Andriy M; Doliba, Nataliya M; Wehrli, Suzanne L; Osbakken, Mary D

    2015-03-01

    Adenylate kinase plays an important role in cellular energy homeostasis by catalysing the interconversion of adenine nucleotides. The goal of present study was to evaluate the contribution of the adenylate kinase reaction to oxidative ATP synthesis by direct measurements of ATP using (31) P NMR spectroscopy. Results show that AMP can stimulate ATP synthesis in the presence or absence of ADP. In particular, addition of 1 mM AMP to the 0.6 mM ADP superfusion system of isolated superfused mitochondria (contained and maintained in agarose beads) led to a 25% increase in ATP synthesis as measured by the increase in βATP signal. More importantly, we show that AMP can support ATP synthesis in the absence of ADP, demonstrated as follows. Superfusion of mitochondria without ADP led to the disappearance of ATP γ, α and β signals and the increase of Pi . Addition of AMP to the medium restored the production of ATP, as demonstrated by the reappearance of γ, α and β ATP signals, in conjunction with a decrease in Pi , which is being used for ATP synthesis. Polarographic studies showed Mg(2+) dependence of this process, confirming the specificity of the adenylate kinase reaction. Furthermore, data obtained from this study demonstrate, for the first time, that different aspects of the adenylate kinase reaction can be evaluated with (31) P NMR spectroscopy. SIGNIFICANCE OF RESEARCH PARAGRAPH: The data generated in the present study indicate that (31) P NMR spectroscopy can effectively be used to study the adenylate kinase reaction under a variety of conditions. This is important because understanding of adenylate kinase function and/or malfunction is essential to understanding its role in health and disease. The data obtained with (31) P NMR were confirmed by polarographic studies, which further strengthens the robustness of the NMR findings. In summary, (31) P NMR spectroscopy provides a sensitive tool to study adenylate kinase activity in different physiological and

  13. Freeze-thaw effects on metabolic enzymes in wood frog organs.

    PubMed

    Cowan, K J; Storey, K B

    2001-08-01

    To determine whether episodes of natural freezing and thawing altered the metabolic makeup of wood frog (Rana sylvatica) organs, the maximal activities of 28 enzymes of intermediary metabolism were assessed in six organs (brain, heart, kidney, liver, skeletal muscle, gut) of control (5 degrees C acclimated), frozen (24 h at -3 degrees C), and thawed (24 h back at 5 degrees C) frogs. The enzymes assessed represented pathways including glycolysis, gluconeo-genesis, amino acid metabolism, fatty acid metabolism, the TCA cycle, and adenylate metabolism. Organ-specific responses seen included (a) the number of enzymes affected by freeze-thaw (1 in gut ranging to 17 in heart), (b) the magnitude and direction of response (most often enzyme activities decreased during freezing and rebounded with thawing but, liver showed freeze-specific increases in several enzymes), and (c) the response to freezing versus thawing (enzyme activities in gut and kidney changed during freezing, whereas most enzymes in skeletal muscle responded to thawing). Overall, the data show that freeze-thaw implements selected changes to the maximal activities of various enzymes of intermediary metabolism and that these may aid organ-specific responses that alter fuel use during freeze-thaw, support cryoprotectant metabolism, and aid organ endurance of freeze-induced ischemia.

  14. Knocking down the expression of adenylate cyclase-associated protein 1 inhibits the proliferation and migration of breast cancer cells.

    PubMed

    Yu, Xia-Fei; Ni, Qi-Chao; Chen, Jin-Peng; Xu, Jun-Fei; Jiang, Ying; Yang, Shu-Yun; Ma, Jing; Gu, Xiao-Ling; Wang, Hua; Wang, Ying-Ying

    2014-04-01

    Adenylate cyclase-associated protein 1 (CAP1) is a conserved protein that was found to be up-regulated in breast cancer and related to the migration of breast cancer. We verified its roles in breast cancer specimens and cell lines. In our results, 71 of 100 specimens of breast cancer showed high levels of CAP1 by immunohistochemistry. Associated with statistical analysis, we saw that CAP1 was related to the grade of breast cancer. In MDA-MB-231, the expression of CAP1 was the highest and by knocking down the expression of CAP1 in MDA-MB-231, its ability for proliferating and migrating apparently decreased and induced changes in morphology, which were related to the arrangement of F-actin. Therefore, CAP1 might be a potential molecular targeted therapy for surgery and immune treatment.

  15. Inhibition of human platelet adenylate cyclase activity by adrenaline, thrombin and collagen: analysis and reinterpretation of experimental data.

    PubMed Central

    Juska, A; Farndale, R W

    1999-01-01

    Mathematical models based on the current understanding of stimulation and inhibition of adenylate cyclase (AC) activity have been developed and used to analyse experimental data [Farndale, Winkler, Martin and Barnes (1992) Biochem. J. 282, 2532] describing the inhibition of human platelet AC by collagen, thrombin and adrenaline. Here it has been demonstrated that neither affinities of receptors specific for adrenaline or thrombin nor the activity of cAMP phosphodiesterase are affected by collagen. Both collagen and thrombin at high doses act as effective inhibitors of AC activity. Inhibition of AC activity by collagen proceeds via two parallel pathways; the same is true for thrombin at moderate concentrations, and the two ligands act independently. The G-protein-dependence of these pathways is distinct from that mediating inhibition of AC activity by adrenaline, i.e. Gi2. Convergence of the inhibitory pathways takes place at the catalytic subunit of AC. PMID:10391837

  16. Cyclic AMP-Mediated Suppression of Neutrophil Extracellular Trap Formation and Apoptosis by the Bordetella pertussis Adenylate Cyclase Toxin

    PubMed Central

    Gray, Mary C.; Hewlett, Erik L.

    2014-01-01

    The adenylate cyclase toxin (ACT) of Bordetella pertussis intoxicates target cells by generating supraphysiologic levels of intracellular cyclic AMP (cAMP). Since ACT kills macrophages rapidly and potently, we asked whether ACT would also kill neutrophils. In fact, ACT prolongs the neutrophil life span by inhibiting constitutive apoptosis and preventing apoptosis induced by exposure to live B. pertussis. Imaging of B. pertussis-exposed neutrophils revealed that B. pertussis lacking ACT induces formation of neutrophil extracellular traps (NETs), whereas wild-type B. pertussis does not, suggesting that ACT suppresses NET formation. Indeed, ACT inhibits formation of NETs by generating cAMP and consequently inhibiting the oxidative burst. Convalescent-phase serum from humans following clinical pertussis blocks the ACT-mediated suppression of NET formation. These studies provide novel insight into the phagocyte impotence caused by ACT, which not only impairs neutrophil function but also inhibits death of neutrophils by apoptosis and NETosis. PMID:25287922

  17. Systematic analysis of gene expression alterations and clinical outcomes of adenylate cyclase-associated protein in cancer

    PubMed Central

    Xie, Shuanshuan; Shen, Changxing; Tan, Min; Li, Ming; Song, Xiaolian; Wang, Changhui

    2017-01-01

    Adenylate Cyclase-associated protein (CAP) is an evolutionarily conserved protein that regulates actin dynamics. Our previous study indicates that CAP1 is overexpressed in NSCLC tissues and correlated with poor clinical outcomes, but CAP1 in HeLa cells actually inhibited migration and invasion, the role of CAP was discrepancy in different cancer types. The present study aims to determine whether CAP can serve as a prognostic marker in human cancers. The CAP expression was assessed using Oncomine database to determine the gene alteration during carcinogenesis, the copy number alteration, or mutations of CAP using cBioPortal, International Cancer Genome Consortium, and Tumorscape database investigated, and the association between CAP expression and the survival of cancer patient using Kaplan-Meier plotter and PrognoScan database evaluated. Therefore, the functional correlation between CAP expression and cancer phenotypes can be established; wherein CAP might serve as a diagnostic marker or therapeutic target for certain types of cancers. PMID:28423713

  18. Pituitary adenylate cyclase activating polypeptide in stress-related disorders: data convergence from animal and human studies.

    PubMed

    Hammack, Sayamwong E; May, Victor

    2015-08-01

    The maladaptive expression and function of several stress-associated hormones have been implicated in pathological stress and anxiety-related disorders. Among these, recent evidence has suggested that pituitary adenylate cyclase activating polypeptide (PACAP) has critical roles in central neurocircuits mediating stress-related emotional behaviors. We describe the PACAPergic systems, the data implicating PACAP in stress biology, and how altered PACAP expression and signaling may result in psychopathologies. We include our work implicating PACAP signaling within the bed nucleus of the stria terminalis in mediating the consequences of stressor exposure and relatedly, describe more recent studies suggesting that PACAP in the central nucleus of the amygdala may impact the emotional aspects of chronic pain states. In aggregate, these results are consistent with data suggesting that PACAP dysregulation is associated with posttraumatic stress disorder in humans. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  19. Phorbol esters alter adenylate cyclase responses to vasoactive intestinal peptide and forskolin in the GH cell line

    SciTech Connect

    Summers, S.; Florio, T.; Cronin, M.

    1986-05-01

    Activation of protein kinase C with phorbol ester modifies cyclic AMP production in several anterior pituitary cell systems. In the GH cell line from a rat pituitary tumor, exposure to phorbol 12-myristate 13-acetate (PMA: 100 nM) for 30 minutes significantly reduces vasoactive intestinal peptide (VIP: 100 nM) stimulated adenylate cyclase (AC) activity in subsequent membrane preparations to 62 + 4% of control (n = 6 independent studies). In contrast, these same membrane preparations respond to forskolin (1 ..mu..M) with significantly more activity, 130 +/- 6% of controls (n = 6 independent studies). Finally, phorbol ester does not block an inhibitory hormone input into the AC system; somatostatin (100 nM) reduction of VIP-stimulated AC activity is not significantly different in membrane preparations from PMA treated and control cells (n = 3 independent studies). These other findings lead the authors to propose that protein kinase C can modify several sites in the AC complex in anterior pituitary cells.

  20. Effects of cimetidine on adenylate cyclase activity of guinea pig gastric mucosa stimulated by histamine, sodium fluoride and 5'-guanylylimidodiphosphate.

    PubMed

    Anttila, P; Westermann, E

    1976-08-01

    Cimetidine, a recently developed histamine H2-receptor blocking agent has been shown to be a potent inhibitor of histamine-stimulated gastric acid secretion in rat, cat, dog and man. To study the mode of action of cimetidine the modification of stimulatory effects of histamine, sodium flouride and 5'-guanylylimidodiphosphate by cimetidine on the adenylate cyclase activity of guinea pig gastric mucosa was studied. The effect of cimetidine was also compared to that of metiamide, an older histamine H2-receptor antagonist. The effect of cimetidine was qualitatively similar to that of metiamide, i.e. a selective blockade of histamine H2-receptors. Quantitatively cimetidine was about 10-fold more potent than metiamide.

  1. Pituitary adenylate cyclase activating polypeptide in stress-related disorders: data convergence from animal and human studies

    PubMed Central

    May, Victor

    2014-01-01

    The maladaptive expression and function of several stress-associated hormones have been implicated in pathological stress- and anxiety-related disorders. Among these, recent evidence has suggested that pituitary adenylate cyclase activating polypeptide (PACAP) has critical roles in central neurocircuits mediating stress-related emotional behaviors. We describe the PACAPergic systems, the data implicating PACAP in stress biology and how altered PACAP expression and signaling may result in psychopathologies. We include our work implicating PACAP signaling within the bed nucleus of the stria terminalis (BNST) in mediating the consequences of stressor exposure and relatedly, describe more recent studies suggesting that PACAP in the central nucleus of the amygdala (CeA) may impact the emotional aspects of chronic pain states. In aggregate, these results are consistent with data suggesting that PACAP dysregulation is associated with post-traumatic stress disorder (PTSD) in humans. PMID:25636177

  2. Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5'-triphosphate.

    PubMed

    Guzmán Luna, Carolina; Costán-Longares, Ana; Lucena, Francisco; Jofre, Joan

    2009-10-01

    The feasibility of detecting somatic coliphages by phage infection of Escherichia coli WG5 and measurement of phage propagation by the lysis mediated release of the bacterial host adenylate kinase (AK) and adenosine 5'-triphosphate (ATP) detected by a bioluminescent signal was evaluated. After 2h of incubation, all cultures infected with reference bacteriophage phiX174 showed a significant increase in the bioluminescent signal, even with number of phages as low as less of 10 plaque forming units (PFU). Naturally occurring somatic coliphages ensured a significant bioluminescent signal after 3h of infection when >10 PFU were inoculated. These results indicate that an easy and reliable method to detect low numbers of coliphages in less than 3h is feasible.

  3. Roles of Protein Kinase A and Adenylate Cyclase in Light-Modulated Cellulase Regulation in Trichoderma reesei

    PubMed Central

    Schuster, André; Tisch, Doris; Seidl-Seiboth, Verena; Kubicek, Christian P.

    2012-01-01

    The cyclic AMP (cAMP) pathway represents a central signaling cascade with crucial functions in all organisms. Previous studies of Trichoderma reesei (anamorph of Hypocrea jecorina) suggested a function of cAMP signaling in regulation of cellulase gene expression. We were therefore interested in how the crucial components of this pathway, adenylate cyclase (ACY1) and cAMP-dependent protein kinase A (PKA), would affect cellulase gene expression. We found that both ACY1 and PKA catalytic subunit 1 (PKAC1) are involved in regulation of vegetative growth but are not essential for sexual development. Interestingly, our results showed considerably increased transcript abundance of cellulase genes in darkness compared to light (light responsiveness) upon growth on lactose. This effect is strongly enhanced in mutant strains lacking PKAC1 or ACY1. Comparison to the wild type showed that ACY1 has a consistently positive effect on cellulase gene expression in light and darkness, while PKAC1 influences transcript levels of cellulase genes positively in light but negatively in darkness. A function of PKAC1 in light-modulated cellulase gene regulation is also reflected by altered complex formation within the cel6a/cbh2 promoter in light and darkness and in the absence of pkac1. Analysis of transcript levels of cellulase regulator genes indicates that the regulatory output of the cAMP pathway may be established via adjustment of XYR1 abundance. Consequently, both adenylate cyclase and protein kinase A are involved in light-modulated cellulase gene expression in T. reesei and have a dampening effect on the light responsiveness of this process. PMID:22286997

  4. Properties of rat anterior pituitary vasopressin receptors: relation to adenylate cyclase and the effect of corticotropin-releasing factor.

    PubMed Central

    Gaillard, R C; Schoenenberg, P; Favrod-Coune, C A; Muller, A F; Marie, J; Bockaert, J; Jard, S

    1984-01-01

    Crude plasma membrane fractions were prepared from female Wistar rat anterior pituitaries. These fractions contained a single population of specific 3H-labeled [8-lysine]vasopressin [( 3H]vasopressin) binding sites with a dissociation of constant (Kd) of 8 +/- 2 X 10(-9) M and maximal binding capacity of 244 +/- 45 fmol/mg of protein. The Kd values for a series of vasopressin structural analogues with selective vasopressor or antidiuretic activities were determined together with the corresponding corticotropin-releasing activities (isolated perfused pituitary cells were used). A good correspondence was found between the two sets of values, suggesting that the detected vasopressin binding sites are the receptors involved in vasopressin-induced corticotropin release. The order of potency of these analogues for the binding to hypophysial receptors was similar to that found for the binding to the receptors involved in the vasopressor response. Corticotropin-releasing factor and angiotensin did not affect vasopressin binding to pituitary membranes. Median eminence extracts inhibited [3H]vasopressin binding with an efficiency very close to that expected from their vasopressin content. Corticotropin-releasing factor activated, and angiotensin inhibited, the adenylate cyclase activity of pituitary membranes. Under the same experimental conditions, vasopressin did not influence adenylate cyclase activity nor did it affect the corticotropin-releasing factor-induced activation. These data support the view that vasopressin is one component of the multifactorial regulation of corticotropin release and that it acts through a cAMP-independent pathway. The potentiation by vasopressin of corticotropin-releasing factor-induced cAMP accumulation in intact cells very likely proceeds through indirect mechanisms, which are not expressed in broken cell preparations. PMID:6326152

  5. Elevated Liver Enzymes

    MedlinePlus

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  6. Kelch-repeat proteins interacting with the Gα protein Gpa2 bypass adenylate cyclase for direct regulation of protein kinase A in yeast

    PubMed Central

    Peeters, Tom; Louwet, Wendy; Geladé, Ruud; Nauwelaers, David; Thevelein, Johan M.; Versele, Matthias

    2006-01-01

    The cAMP–PKA pathway consists of an extracellular ligand-sensitive G protein-coupled receptor, a G protein signal transmitter, and the effector, adenylate cyclase, of which the product, cAMP, acts as an intracellular second messenger. cAMP activates PKA by dissociating the regulatory subunit from the catalytic subunit. Yeast cells (Saccharomyces cerevisiae) contain a glucose/sucrose-sensitive seven-transmembrane domain receptor, Gpr1, that was proposed to activate adenylate cyclase through the Gα protein Gpa2. Consistently, we show here that adenylate cyclase binds only to active, GTP-bound Gpa2. Two related kelch-repeat proteins, Krh1/Gpb2 and Krh2/Gpb1, are associated with Gpa2 and were suggested to act as Gβ mimics for Gpa2, based on their predicted seven-bladed β-propeller structure. However, we find that although Krh1 associates with both GDP and GTP-bound Gpa2, it displays a preference for GTP-Gpa2. The strong down-regulation of PKA targets by Krh1 and Krh2 does not require Gpa2 but is strictly dependent on both the catalytic and the regulatory subunits of PKA. Krh1 directly interacts with PKA by means of the catalytic subunits, and Krh1/2 stimulate the association between the catalytic and regulatory subunits in vivo. Indeed, both a constitutively active GPA2 allele and deletion of KRH1/2 lower the cAMP requirement of PKA for growth. We propose that active Gpa2 relieves the inhibition imposed by the kelch-repeat proteins on PKA, thereby bypassing adenylate cyclase for direct regulation of PKA. Importantly, we show that Krh1/2 also enhance the association between mouse R and C subunits, suggesting that Krh control of PKA has been evolutionarily conserved. PMID:16924114

  7. Studies on responsiveness of hepatoma cells to catecholamines. III. Difference between the receptor-adenylate cyclase regulating systems in AH130 cells and cultured normal rat liver cells.

    PubMed

    Sanae, F; Matsunaga, T; Miyamoto, K; Koshiura, R

    1986-10-01

    The responsiveness to three beta-adrenergic agonists, isoproterenol (IPN), epinephrine (Epi) and norepinephrine (NE) in AH13O cells was examined compared with that in normal rat liver cells which were cultured for 24 hr after collagenase digestion. As regards to the activation of adenylate cyclase in the cell homogenates, the relative affinity of the three agonists was in order of IPN greater than NE greater than Epi in AH130 cells and IPN greater than Epi greater than NE in cultured normal liver cells. While the efficacies of the three agonists were similar in cultured liver cells, those of NE and Epi were markedly lower than that of IPN in AH13O cells and were increased to the similar level of IPN by pretreatment with phentolamine, but not with prazosin. Clonidine inhibited the activation of adenylate cyclase by IPN in AH13O cells. When cells were preincubated with islet-activating protein (IAP), the activity of adenylate cyclase in the presence or absence of agonist in both cell lines increased. In IAP-treated AH13O cells, the efficacies of NE and Epi became close to that of IPN. Adenylate cyclase in IAP-treated AH13O cells was activated by GTP in a dose-dependent manner, but that in IAP-treated cultured liver cells was not. In the presence of IPN, biphasic (activatory and inhibitory) effects of GTP on the cyclase were observed, and the inhibitory phase was eliminated by the IAP-treatment in both cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Human 5-HT7 receptor-induced inactivation of forskolin-stimulated adenylate cyclase by risperidone, 9-OH-risperidone and other "inactivating antagonists".

    PubMed

    Toohey, Nicole; Klein, Michael T; Knight, Jessica; Smith, Carol; Teitler, Milt

    2009-09-01

    We have previously reported on the unusual human 5-hydroxytryptamine(7) (h5-HT(7)) receptor-inactivating properties of risperidone, 9-OH-risperidone, bromocriptine, methiothepin, metergoline, and lisuride. Inactivation was defined as the inability of 10 microM 5-HT to stimulate cAMP accumulation after brief exposure and thorough removal of the drugs from HEK293 cells expressing h5-HT(7) receptors. Herein we report that brief exposure of the h5-HT(7) receptor-expressing cells to inactivating drugs, followed by removal of the drugs, results in potent and efficacious irreversible inhibition of forskolin-stimulated adenylate cyclase activity. Pretreatment, followed by removal of the inactivating drugs inhibited 10 microM forskolin-stimulated adenylate cyclase activity with potencies similar to the drugs' affinities for the h5-HT(7) receptor. The actions of the inactivating drugs were pertussis toxin-insensitive, indicating the lack of G(i) in their mechanism(s) of action. Methiothepin and bromocriptine maximally inhibited 10 microM forskolin-stimulated adenylate cyclase, whereas the other drugs produced partial inhibition, indicating the drugs are inducing slightly different inactive conformations of the h5-HT(7) receptor. Maximal effects of these inactivating drugs occurred within 15 to 30 min of exposure of the cells to the drugs. A G(s)-mediated inhibition of forskolin-stimulated activity has never been reported. The inactivating antagonists seem to induce a stable conformation of the h5-HT(7) receptor, which induces an altered state of G(s), which, in turn, inhibits forskolin-mediated stimulation of adenylate cyclase. These and previous observations indicate that the inactivating antagonists represent a unique class of drugs and may reveal GPCR regulatory mechanisms previously unknown. These drugs may produce innovative approaches to the development of therapeutic drugs.

  9. Characterization of the 5-hydroxytryptamine1a receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes.

    PubMed

    De Vivo, M; Maayani, S

    1986-07-01

    The inhibition of forskolin-stimulated adenylate cyclase activity by 5-hydroxytryptamine (5-HT) receptor agonists was measured in guinea pig and rat hippocampal membranes. The results were consistent with the inhibition being mediated by a single, homogeneous population of receptors. In guinea pig hippocampal membranes 8-hydroxy-2-(di-n-propylamino)tetralin, d-lysergic acid diethylamide, 5-HT and buspirone were potent in inhibiting forskolin-stimulated adenylate cyclase activity, with EC50 values of 18, 24, 53 and 146 nM, respectively. Spiperone (Kb = 26 nM) and methiothepin (Kb = 13 nM) were potent competitive antagonists at this receptor whereas ketanserin, a high affinity 5-HT2 receptor ligand, and ICS 205-930, a high affinity peripheral neuronal (M) receptor ligand, were not. In rat hippocampal membranes, 8-hydroxy-2-(di-n-propylamino)tetralin, d-lysergic acid diethylamide, 5-HT and buspirone were potent agonists and exhibited the same rank order of potency as in guinea pig hippocampal membranes. The maximal percentage of inhibition by buspirone was significantly less than the maximal percentage of inhibition by 5-HT in rat membranes, suggesting that it is a partial agonist at this receptor, with an intrinsic activity relative to 5-HT of 0.5. The concentration-response data show that the inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes is mediated by a receptor with the characteristics of the 5-HT1A binding site. We propose that the inhibition of adenylate cyclase activity is a functional correlate of this binding site. This response is suitable for measuring activities and affinities of drugs acting at 5-HT1A receptors.

  10. Fundamentals of enzyme kinetics.

    PubMed

    Seibert, Eleanore; Tracy, Timothy S

    2014-01-01

    This chapter provides a general introduction to the kinetics of enzyme-catalyzed reactions, with a focus on drug-metabolizing enzymes. A prerequisite to understanding enzyme kinetics is having a clear grasp of the meanings of "enzyme" and "catalysis." Catalysts are reagents that can increase the rate of a chemical reaction without being consumed in the reaction. Enzymes are proteins that form a subset of catalysts. These concepts are further explored below.

  11. Corticotropin-releasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'-monophosphate system

    SciTech Connect

    Dave, J.R.; Eiden, L.E.; Eskay, R.L.

    1985-06-01

    Specific binding sites for rat corticotropin-releasing factor (rCRF) are present in rat adrenal medulla, ventral prostate, spleen, liver, kidney, and testis and bovine chromaffin cells in culture. Maximal binding of (/sup 125/I)rCRF occurred within 25 min at 4 C and was saturable. Scatchard analysis of rCRF binding to rat adrenal membranes and bovine chromaffin cells revealed the existence of two classes of binding sites. One class had a relatively higher apparent affinity and lower number of binding sites, whereas the other class had a relatively lower affinity and higher number of binding sites. CRF induced a dose-related increase in rat adrenal membrane adenylate cyclase activity and cAMP levels in bovine chromaffin cells. Nanomolar concentrations of rCRF maximally stimulated adenylate cyclase activity in rat adrenal membranes and maximally increased cAMP levels in bovine chromaffin cells to 86% and 130% above control values, respectively. The demonstration of specific CRF-binding sites in a variety of peripheral tissues and the finding that activation of specific CRF-binding sites in adrenal tissue stimulates the adenylate cyclase-cAMP system suggest that CRF may have an important regulatory role in various peripheral tissues.

  12. Cardiovascular and adenylate cyclase stimulating effects of colforsin daropate, a water-soluble forskolin derivative, compared with those of isoproterenol, dopamine and dobutamine.

    PubMed

    Yoneyama, Masahiko; Sugiyama, Atsushi; Satoh, Yoshioki; Takahara, Akira; Nakamura, Yuji; Hashimoto, Keitaro

    2002-12-01

    Colforsin daropate is a recently developed water-soluble derivative of forskolin that directly stimulates adenylate cyclase, unlike the catecholamines. The chronotropic, inotropic and coronary vasodilator actions of colforsin daropate were compared with those of isoproterenol, dopamine and dobutamine, using canine isolated, blood-perfused heart preparations. The stimulating effect of each drug on adenylate cyclase activity was also assessed. Colforsin daropate, as well as each of the catecholamines, exerted positive chronotropic, inotropic and coronary vasodilator actions. The order of selectivity for the cardiovascular variables of colforsin daropate was coronary vasodilation > positive inotropy > positive chronotropy; whereas that of isoproterenol, dopamine and dobutamine was positive inotropy > coronary vasodilation > positive chronotropy. Thus, a marked characteristic of colforsin daropate is its potent coronary vasodilator action. On the other hand, each drug significantly increased the adenylate cyclase activity in a dose-related manner: colforsin daropate > isoproterenol > dopamine = dobutamine. These results suggest that colforsin daropate may be preferable in the treatment of severe heart failure where the coronary blood flow is reduced and beta-adrenoceptor-dependent signal transduction pathway is down-regulated.

  13. Modulation of replication, aminoacylation and adenylation in vitro and infectivity in vivo of BMV RNAs containing deletions within the multifunctional 3' end.

    PubMed Central

    Bujarski, J J; Ahlquist, P; Hall, T C; Dreher, T W; Kaesberg, P

    1986-01-01

    The genome of brome mosaic virus (BMV) is comprised of three (+) strand RNAs, each containing a similar, highly structured, 200 base long sequence at its 3' end. A 134 base subset of this sequence contains signals directing interaction of the viral RNA with BMV RNA replicase, ATP,CTP:tRNA nucleotidyl transferase and aminoacyl tRNA synthetase. A series of mutants containing deletions within this region, previously constructed and tested in vitro for the effect on replication and aminoacylation activities, has now been assayed in vitro for adenylation function and in vivo for ability to replicate in isolated protoplasts and whole plants. These tests indicate that features of viral RNA recognized by BMV replicase overlap those directing adenylation, but are distinct from those directing aminoacylation. Consequently, the lethality of a deletion preferentially inhibiting aminoacylation suggests that this function may have an essential role contributing to viral replication in vivo. An RNA3 mutant bearing a 20-base deletion yielding normal levels of aminoacylation and enhanced levels of replicase template activity and adenylation in vitro was able to replicate in protoplasts and plants; however, its accumulation in protoplasts was reduced relative to wild-type. This suggests that additional functions affecting the replication and accumulation of viral RNA reside in the conserved 3' sequence. Images Fig. 2. Fig. 3. Fig. 4. PMID:3758026

  14. A New Type of Metal-Binding Site in Cobalt- And Zinc-Containing Adenylate Kinases Isolated From Sulfate-Reducers D. Gigas And D. Desulfuricans ATCC 27774

    SciTech Connect

    Gavel, O.Y.; Bursakov, S.A.; Rocco, G.Di; Trincao, J.; Pickering, I.J.; George, G.N.; Calvete, J.J.; Brondino, C.; Pereira, A.S.; Lampreia, J.; Tavares, P.; Moura, J.J.G.; Moura, I.

    2009-05-18

    Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterized in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the 'LID' domain. The sequence {sup 129}Cys-X{sub 5}-His-X{sub 15}-Cys-X{sub 2}-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

  15. A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies.

    PubMed

    Jayakumar, Thanasekaran; Chen, Wei-Fan; Lu, Wan-Jung; Chou, Duen-Suey; Hsiao, George; Hsu, Chung-Yi; Sheu, Joen-Rong; Hsieh, Cheng-Ying

    2013-06-01

    Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15-75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca(2+) mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH(●)) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH(●) formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This

  16. Illuminating the Mechanistic Roles of Enzyme Conformational Dynamics

    SciTech Connect

    Hanson, Jeffrey A.; Dunderstadt, Karl; Watkins, Lucas P.; Bhattacharyya, Sucharita; Brokaw, Jason B.; Chu, Jhih-wei; Yang, Haw

    2007-11-13

    Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme’s lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK’s domain movements on its catalytic time scale. To quantitatively measure the enzyme’s entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme’s conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme’s rate-limiting step.

  17. Secretion of adenylate kinase 1 is required for extracellular ATP synthesis in C2C12 myotubes

    PubMed Central

    Choo, Hyo-Jung; Kim, Bong-Woo; Kwon, Oh-Bong; Lee, Chang Seok; Choi, Jong-Soon

    2008-01-01

    Extracellular ATP (exATP) has been known to be a critical ligand regulating skeletal muscle differentiation and contractibility. ExATP synthesis was greatly increased with the high level of adenylate kinase 1 (AK1) and ATP synthase β during C2C12 myogenesis. The exATP synthesis was abolished by the knock-down of AK1 but not by that of ATP synthase β in C2C12 myotubes, suggesting that AK1 is required for exATP synthesis in myotubes. However, membrane-bound AK1β was not involved in exATP synthesis because its expression level was decreased during myogenesis in spite of its localization in the lipid rafts that contain various kinds of receptors and mediate cell signal transduction, cell migration, and differentiation. Interestingly, cytoplasmic AK1 was secreted from C2C12 myotubes but not from C2C12 myoblasts. Taken together all these data, we can conclude that AK1 secretion is required for the exATP generation in myotubes. PMID:18446060

  18. Studies of the cAMP mediated aggregation in Dictyostelium discoideum: receptor mediated activation of the adenylate cyclase

    SciTech Connect

    Theibert, W.E.A.B.

    1985-01-01

    Dictyostelium discoideum, a eukaryotic amoeba of the cellular slime mold family, provides an interesting paradigm in developmental biology. During development, hundreds of thousands of cells aggregate to form a multicellular aggregate. Aggregation is mediated by chemotaxis and chemical signaling. Waves of adenosine 3'-5' cyclic monophosphate (cAMP) propagate through the monolayer and provide transient gradients for chemotaxis. The author has used a reversible inhibitor of the cAMP signaling response to demonstrate that adaptation to cAMP is independent of the activation of the adenylate cyclase and therefore is not caused by the rise in intracellular cAMP. Next, it is shown that adenosine inhibits the cAMP signaling response. Inhibition is rapid, reversible, and depends on the cAMP stimulus concentration. Then the specificity of the cAMP receptors which mediates signaling is determined and compared with the receptors which mediate chemotaxis, the cGMP response, and cAMP binding antagonism. The cAMP surface receptor has been identified by photoaffinity labeling intact cells with (/sup 32/P)-8-N/sub 3/-cAMP using an ammonium sulfate binding stabilization technique. The photoactivated ligand specifically labels a polypeptide, localized to the membrane fraction, which migrates as a closely spaced doublet on SDS Page.

  19. Stress-related disorders, pituitary adenylate cyclase-activating peptide (PACAP)ergic system, and sex differences.

    PubMed

    Ramikie, Teniel S; Ressler, Kerry J

    2016-12-01

    Trauma-related disorders, such as posttraumatic stress disorder (PTSD) are remarkably common and debilitating, and are often characterized by dysregulated threat responses. Across numerous epidemiological studies, females have been found to have an approximately twofold increased risk for PTSD and other stress-related disorders. Understanding the biological mechanisms of this differential risk is of critical importance. Recent data suggest that the pituitary adenylate cyclase-activating polypeptide (PACAP) pathway is a critical regulator of the stress response across species. Moreover, increasing evidence suggests that this pathway is regulated by both stress and estrogen modulation and may provide an important window into understanding mechanisms of sex differences in the stress response. We have recently shown that PACAP and its receptor (PAC1R) are critical mediators of abnormal processes after psychological trauma. Notably, in heavily traumatized human subjects, there appears to be a robust sex-specific association of PACAP blood levels and PAC1R gene variants with fear physiology, PTSD diagnosis, and symptoms, specifically in females. The sex-specific association occurs within a single-nucleotide polymorphism (rs2267735) that resides in a putative estrogen response element involved in PAC1R gene regulation. Complementing these human data, the PAC1R messenger RNA is induced with fear conditioning or estrogen replacement in rodent models. These data suggest that perturbations in the PACAP-PAC1R pathway are regulated by estrogen and are involved in abnormal fear responses underlying PTSD.

  20. Pharmacological characterization of the dopamine-sensitive adenylate cyclase in cockroach brain: evidence for a distinct dopamine receptor

    SciTech Connect

    Orr, G.L.; Gole, J.W.D.; Notman, H.J.; Downer, R.G.H.

    1987-12-21

    Dopamine increases cyclic AMP production in crude membrane preparations of cockroach brain with plateaus in cyclic AMP production occurring between 1-10 ..mu..M and 10 mM. Maximal production of cyclic AMP is 2.25 fold greater than that of control values. Octopamine also increases cyclic AMP production with a Ka of 1.4 ..mu..M and maximal production 3.5 fold greater than that of control. 5-Hydroxytryptamine does not increase cyclic AMP production. The effects of octopamine and dopamine are fully additive. The vertebrate dopamine agonists ADTN and epinine stimulate the dopamine-sensitive adenylate cyclase (AC) with Ka values of 4.5 and 0.6 ..mu..M respectively and with maximal effectiveness 1.7 fold greater than that of control. The selective D/sub 2/-dopamine agonist LY-171555 stimulates cyclic AMP production to a similar extent with a Ka of 50 ..mu..M. Other dopamine agonists have no stimulatory effects. With the exception of mianserin, /sup 3/H-piflutixol is displaced from brain membranes by dopamine antagonists with an order of potency similar to that observed for the inhibition of dopamine-sensitive AC. The results indicate that the octopamine- and dopamine-sensitive AC in cockroach brain can be distinguished pharmacologically and the dopamine receptors coupled to AC have pharmacological characteristics distinct from vertebrate D/sup 1/- and D/sup 2/-dopamine receptors. 33 references, 3 figures, 2 tables.

  1. Characterization and Engineering of the Adenylation Domain of a NRPS-Like Protein: A Potential Biocatalyst for Aldehyde Generation

    PubMed Central

    2015-01-01

    The adenylation (A) domain acts as the first “gate-keeper” to ensure the activation and thioesterification of the correct monomer to nonribosomal peptide synthetases (NRPSs). Our understanding of the specificity-conferring code and our ability to engineer A domains are critical for increasing the chemical diversity of nonribosomal peptides (NRPs). We recently discovered a novel NRPS-like protein (ATEG_03630) that can activate 5-methyl orsellinic acid (5-MOA) and reduce it to 2,4-dihydroxy-5,6-dimethyl benzaldehyde. A NRPS-like protein is much smaller than multidomain NRPSs, but it still represents the thioesterification half-reaction, which is otherwise missed from a stand-alone A domain. Therefore, a NRPS-like protein may serve as a better model system for A domain engineering. Here, we characterize the substrate specificity of ATEG_03630 and conclude that the hydrogen-bond donor at the 4-position is crucial for substrate recognition. Next, we show that the substrate specificity of ATEG_03630 can be engineered toward our target substrate anthranilate via bioinformatics analysis and mutagenesis. The resultant mutant H358A increased its activity toward anthranilate by 10.9-fold, which led to a 26-fold improvement in specificity. Finally, we demonstrate one-pot chemoenzymatic synthesis of 4-hydroxybenzaldoxime from 4-hydroxybenzoic acid with high yield. PMID:24804152

  2. Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti-Medicago symbiosis.

    PubMed

    Tian, Chang Fu; Garnerone, Anne-Marie; Mathieu-Demazière, Céline; Masson-Boivin, Catherine; Batut, Jacques

    2012-04-24

    Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has triggered the symbiotic plant developmental program. Here we identify in Sinorhizobium meliloti, the Medicago symbiont, a cAMP-signaling regulatory cascade consisting of three receptor-like adenylate cyclases, a Crp-like regulator, and a target gene of unknown function. The cascade is activated specifically by a plant signal during nodule organogenesis. Cascade inactivation results in a hyperinfection phenotype consisting of abortive epidermal infection events uncoupled from nodulation. These findings show that, in response to a plant signal, rhizobia play an active role in the control of infection. We suggest that rhizobia may modulate the plant's susceptibility to infection. This regulatory loop likely aims at optimizing legume infection.

  3. Effectiveness and limitations of local structural entropy optimization in the thermal stabilization of mesophilic and thermophilic adenylate kinases.

    PubMed

    Moon, Sojin; Bannen, Ryan M; Rutkoski, Thomas J; Phillips, George N; Bae, Euiyoung

    2014-10-01

    Local structural entropy (LSE) is a descriptor for the extent of conformational heterogeneity in short protein sequences that is computed from structural information derived from the Protein Data Bank. Reducing the LSE of a protein sequence by introducing amino acid mutations can result in fewer conformational states and thus a more stable structure, indicating that LSE optimization can be used as a protein stabilization method. Here, we describe a series of LSE optimization experiments designed to stabilize mesophilic and thermophilic adenylate kinases (AKs) and report crystal structures of LSE-optimized AK variants. In the mesophilic AK, thermal stabilization by LSE reduction was effective but limited. Structural analyses of the LSE-optimized mesophilic AK variants revealed a strong correlation between LSE and the apolar buried surface area. Additional mutations designed to introduce noncovalent interactions between distant regions of the polypeptide resulted in further stabilization. Unexpectedly, optimizing the LSE of the thermophilic AK resulted in a decrease in thermal stability. This destabilization was reduced when charged residues were excluded from the possible substitutions during LSE optimization. These observations suggest that stabilization by LSE reduction may result from the optimization of local hydrophobic contacts. The limitations of this process are likely due to ignorance of other interactions that bridge distant regions in a given amino acid sequence. Our results illustrate the effectiveness and limitations of LSE optimization as a protein stabilization strategy and highlight the importance and complementarity of local conformational stability and global interactions in protein thermal stability. © 2014 Wiley Periodicals, Inc.

  4. Role of pituitary adenylate cyclase-activating polypeptide in modulating hypothalamus-pituitary neuroendocrine functions in mouse cell models.

    PubMed

    Kanasaki, H; Oride, A; Kyo, S

    2015-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally identified as a hypothalamic activator of cyclic adenosine monophosphate production in pituitary cells. PACAP and its receptor are expressed not only in the central nervous system, but also in peripheral organs, and function to stimulate pituitary hormone synthesis and secretion as both a hypothalamic-pituitary-releasing factor and an autocrine-paracrine factor within the pituitary. PACAP stimulates the expression of the gonadotrophin α, luteinising hormone (LH) β and follicle-stimulating hormone (FSH) β subunits, as well as the gonadotrophin-releasing hormone (GnRH) receptor and its own PACAP type I receptor (PAC1R) in gonadotrophin-secreting pituitary cells. In turn, GnRH, which is known to be a crucial component of gonadotrophin secretion, stimulates the expression of PACAP and PAC1R in gonadotrophs. In addition, PAC1R and PACAP modulate the functions of GnRH-producing neurones in the hypothalamus. This review summarises the current understanding of the possible roles of PACAP and PAC1R in modulating hypothalamus and pituitary neuroendocrine cells in the mouse models.

  5. Stress-related disorders, pituitary adenylate cyclase—activating peptide (PACAP)ergic system, and sex differences

    PubMed Central

    Ramikie, Teniel S.; Ressler, Kerry J.

    2016-01-01

    Trauma-related disorders, such as posttraumatic stress disorder (PTSD) are remarkably common and debilitating, and are often characterized by dysregulated threat responses. Across numerous epidemiological studies, females have been found to have an approximately twofold increased risk for PTSD and other stress-related disorders. Understanding the biological mechanisms of this differential risk is of critical importance. Recent data suggest that the pituitary adenylate cyclase-activating polypeptide (PACAP) pathway is a critical regulator of the stress response across species. Moreover, increasing evidence suggests that this pathway is regulated by both stress and estrogen modulation and may provide an important window into understanding mechanisms of sex differences in the stress response. We have recently shown that PACAP and its receptor (PAC1R) are critical mediators of abnormal processes after psychological trauma. Notably, in heavily traumatized human subjects, there appears to be a robust sex-specific association of PACAP blood levels and PAC1R gene variants with fear physiology, PTSD diagnosis, and symptoms, specifically in females. The sex-specific association occurs within a single-nucleotide polymorphism (rs2267735) that resides in a putative estrogen response element involved in PAC1R gene regulation. Complementing these human data, the PAC1R messenger RNA is induced with fear conditioning or estrogen replacement in rodent models. These data suggest that perturbations in the PACAP-PAC1R pathway are regulated by estrogen and are involved in abnormal fear responses underlying PTSD. PMID:28179812

  6. In vitro toxicity of mercury, cadmium, and arsenic to platelet aggregation: influence of adenylate cyclase and phosphodiesterase activity.

    PubMed

    Kumar, S V; Bhattacharya, S

    2000-01-01

    In vitro effect of mercury (Hg2+), cadmium (Cd2+), and arsenic (As3+) on adenylate cyclase (AC) and phosphodiesterase (PDE) activity in relation to platelet aggregation (PA) was studied in rats. Cd(2+) significantly elevated cAMP (p < 0.005) in a dose-dependent (5, 10 and 20 pmoles) manner while Hg(2+) and As(3+) significantly reduced the cAMP level (p < 0.01 and p < 0.005, respectively). Our studies further reveal that Hg21 and As(3+) inhibit AC and stimulate PDE activity with a concomitant increase in the rate of PA. On the other hand, Cd(2+) stimulates AC and inhibits PDE activity with a decrease in the rate of PA. The present investigation suggests that cellular cAMP is a regulatory molecule in the event of PA and the disruption of its homeostasis is directly correlated to xenobiotic effects on PA. It is concluded that other than divalent heavy metal cations, As(3+) appears to be one of the most toxic xenobiotics to platelet function.

  7. Characterization and Engineering of the Adenylation Domain of a NRPS-Like Protein: A Potential Biocatalyst for Aldehyde Generation.

    PubMed

    Wang, Meng; Zhao, Huimin

    2014-04-04

    The adenylation (A) domain acts as the first "gate-keeper" to ensure the activation and thioesterification of the correct monomer to nonribosomal peptide synthetases (NRPSs). Our understanding of the specificity-conferring code and our ability to engineer A domains are critical for increasing the chemical diversity of nonribosomal peptides (NRPs). We recently discovered a novel NRPS-like protein (ATEG_03630) that can activate 5-methyl orsellinic acid (5-MOA) and reduce it to 2,4-dihydroxy-5,6-dimethyl benzaldehyde. A NRPS-like protein is much smaller than multidomain NRPSs, but it still represents the thioesterification half-reaction, which is otherwise missed from a stand-alone A domain. Therefore, a NRPS-like protein may serve as a better model system for A domain engineering. Here, we characterize the substrate specificity of ATEG_03630 and conclude that the hydrogen-bond donor at the 4-position is crucial for substrate recognition. Next, we show that the substrate specificity of ATEG_03630 can be engineered toward our target substrate anthranilate via bioinformatics analysis and mutagenesis. The resultant mutant H358A increased its activity toward anthranilate by 10.9-fold, which led to a 26-fold improvement in specificity. Finally, we demonstrate one-pot chemoenzymatic synthesis of 4-hydroxybenzaldoxime from 4-hydroxybenzoic acid with high yield.

  8. Rapid, semi-automated, and inexpensive radioimmunoassay of cAMP: application in GPCR-mediated adenylate cyclase assays.

    PubMed

    Brown, Justin T; Kant, Andrew; Mailman, Richard B

    2009-03-15

    Cyclic AMP (cAMP) is an important signal transduction second messenger that is commonly used as a functional mirror on the actions of G protein-coupled receptors that can activate or inhibit adenylate cyclases. A radioimmunoassay for cAMP with femtomole sensitivity was first reported by Steiner more than 30 years ago, and there have been several subsequent modifications that have improved this assay in various ways. Here we describe additional improvement to existing methods that markedly improve speed and reduce cost without sacrificing sensitivity, and is also adaptable to analysis of cGMP. The primary antibody is coupled directly to magnetic beads that are then separated from unbound marker using filtration on microplates. This eliminates the need for a secondary antibody, and markedly increases throughput. In addition, we report a simple, reproducible, and inexpensive method to make the radiomarker used for this assay. Although still requiring the use of radioactivity, the resulting method retains a high degree of accuracy and precision, and is suitable for low-cost high throughput screening. Use of aspects of this method can also improve throughput in other radioimmunoassays.

  9. From endoplasmic reticulum to mitochondria: absence of the Arabidopsis ATP antiporter endoplasmic Reticulum Adenylate Transporter1 perturbs photorespiration.

    PubMed

    Hoffmann, Christiane; Plocharski, Bartolome; Haferkamp, Ilka; Leroch, Michaela; Ewald, Ralph; Bauwe, Hermann; Riemer, Jan; Herrmann, Johannes M; Neuhaus, H Ekkehard

    2013-07-01

    The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO(2) concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants.

  10. Urinary Bladder Dysfunction and Altered Somatic Sensitivity in Pituitary Adenylate Cyclase Activating Polypeptide Knockout (PACAP−/−) Mice

    PubMed Central

    May, Victor; Vizzard, Margaret A.

    2010-01-01

    Purpose Pituitary adenylate cyclase activating polypeptide (PACAP) and receptors are expressed in micturition pathways. Studies demonstrated roles for PACAP in detrusor smooth muscle contraction, facilitating ATP release from urothelium and PACAP antagonism reduced cyclophosphamide-induced bladder hyperreflexia. Materials and Methods PACAP contributions to micturition and somatic sensation were studied in PACAP knockout (PACAP−/−), littermate heterozygote (PACAP+/−) and wildtype (WT) mice using conscious cystometry with continuous intravesical saline or acetic acid (AA; 0.5%) instillation, urination patterns, somatic sensitivity testing of hindpaw and pelvic region with calibrated von Frey filaments and morphological assessments of urinary bladder. Results PACAP−/− mice exhibit increased bladder mass with fewer but larger urine spots. In PACAP−/− mice, the lamina propria and detrusor smooth muscle are significantly thicker whereas the urothelium is unchanged. PACAP−/− mice exhibit increased bladder capacity, void volume (VV) and longer intercontraction interval (ICI) with significantly increased detrusor contraction duration and large residual volume. WT mice respond to AA (0.5%) with a reduction in VV and a decreased ICI whereas PACAP+/− and PACAP−/− mice do not respond. PACAP−/− mice are less responsive to somatic stimulation. PACAP+/− also exhibit bladder dysfunction and somatic and visceral sensory abnormalities but to a lesser degree. Conclusions PACAP gene disruption contributes to changes in bladder morphology, bladder function and somatic and visceral hypoalgesia. PMID:20022034

  11. Pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor is expressed during embryonic development of the earthworm.

    PubMed

    Boros, Akos; Somogyi, Ildikó; Engelmann, Péter; Lubics, Andrea; Reglodi, Dóra; Pollák, Edit; Molnár, László

    2010-03-01

    Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.

  12. Adenylate Cyclase 6 Determines cAMP Formation and Aquaporin-2 Phosphorylation and Trafficking in Inner Medulla

    PubMed Central

    Tang, Tong; Murray, Fiona; Schroth, Jana; Insel, Paul A.; Fenton, Robert A.; Hammond, H. Kirk

    2010-01-01

    Arginine vasopressin (AVP) enhances water reabsorption in the renal collecting duct by vasopressin V2 receptor (V2R)-mediated activation of adenylyl cyclase (AC), cAMP-promoted phosphorylation of aquaporin-2 (AQP2), and increased abundance of AQP2 on the apical membrane. Multiple isoforms of adenylate cyclase exist, and the roles of individual AC isoforms in water homeostasis are not well understood. Here, we found that levels of AC6 mRNA, the most highly expressed AC isoform in the inner medulla, inversely correlate with fluid intake. Moreover, mice lacking AC6 had lower levels of inner medullary cAMP, reduced abundance of phosphorylated AQP2 (at both serine-256 and serine-269), and lower urine osmolality than wild-type mice. Water deprivation or administration of the V2R agonist dDAVP did not increase urine osmolality of AC6-deficient mice to the levels of wild-type mice. Furthermore, AC6-deficient mice lacked dDAVP-promoted inner medullary cAMP formation and phosphorylation of serine-269 and had attenuated increases in both phosphorylation of serine-256 and apical membrane AQP2 trafficking. In summary, AC6 expression determines inner medullary cAMP formation and AQP2 phosphorylation and trafficking, the absence of which causes nephrogenic diabetes insipidus. PMID:20864687

  13. Pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity and mRNA expression in the duck gastrointestinal tract.

    PubMed

    Mirabella, N; Squillacioti, C; Colitti, M; Germano, G; Pelagalli, A; Paino, G

    2002-06-01

    The presence and distribution of pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity were studied in the duck gastrointestinal tract using immunohistochemistry and radioimmunoassays. Expression and distribution of PACAP mRNA were also studied using reverse transcriptase polymerase chain reaction (RT-PCR) and hybridization techniques. In addition, a partial coding sequence (cds) of the duck growth hormone-releasing hormone (GRF)/PACAP gene was identified. The presence of both PACAP-38 and PACAP-27 was demonstrated, the former being the predominant form. PACAP immunoreactivity was found in neurons and fibers of the enteric nervous system (ENS), in endocrine cells and in the gut associated lymphoid tissue (GALT). Double immunostaining showed that PACAP is almost completely colocalized with vasoactive intestinal peptide (VIP) in the ENS. Moreover, PACAP was also found in nitric oxide synthase (NOS)-containing neurons and nerve fibers. Radioimmunoassay (RIA) performed on denervated gut showed that more than one-half of the duodenal PACAP is extrinsic in origin. RT-PCR, Northern blot analysis and in situ hybridization confirmed the immunohistochemical data. The findings of the present study suggest that, in birds, PACAP may have multiple roles in regulating gastrointestinal functions.

  14. Defective dopamine-1 receptor adenylate cyclase coupling in the proximal convoluted tubule from the spontaneously hypertensive rat.

    PubMed Central

    Kinoshita, S; Sidhu, A; Felder, R A

    1989-01-01

    The natriuretic effect of DA-1 agonists is less in the spontaneously hypertensive rat (SHR) than its normotensive control, the Wistar-Kyoto rat (WKY). To determine a mechanism of the decreased effect of DA-1 agonists on sodium transport, DA-1 receptors in renal proximal convoluted tubule (PCT) were studied by radioligand binding and by adenylate cyclase (AC) determinations. Specific binding of 125I-SCH 23982 (defined by 10 microM SCH 23390, a DA-1 antagonist) was concentration dependent, saturable, and stereoselective. The dissociation constant, maximum receptor density, and DA-1 antagonist inhibition constant were similar in SHR and WKY. The apparent molecular weight of the DA-1 receptor determined by the photoaffinity D1 probe 125I-MAB was also similar in WKY and SHR. However, DA-1 agonists competed more effectively for specific 125I-SCH 23982 binding sites in WKY than in SHR. Basal as well as forskolin, parathyroid hormone, GTP and Gpp(NH)p-stimulated-AC activities were similar. In contrast DA-1 agonists (fenoldopam, SKF 38393, SND 911C12) stimulated AC activity to a lesser extent in SHR. GTP and Gpp(NH)p enhanced the ability of DA-1 agonists to stimulate AC activity in WKY but not in SHR. These data suggest a defect in the DA-1 receptor-second messenger coupling mechanism in the PCT of the SHR. Images PMID:2574187

  15. The Nuclear Exosome and Adenylation Regulate Post-Transcriptional Tethering of Yeast GAL genes to the Nuclear Periphery

    PubMed Central

    Vodala, Sadanand; Abruzzi, Katharine Compton; Rosbash, Michael

    2009-01-01

    Some activated yeast genes, including GAL genes, associate with and remain tethered to the nuclear periphery even after transcriptional shutoff. To identify factors that affect GAL gene-nuclear periphery association after transcriptional shutoff, we designed a plasmid-based visual screen. Although many factors affected tethering during transcription, only a few specifically affected post-transcriptional tethering. Two of these, Rrp6p and Lrp1p, are nuclear exosome components with known roles in retaining RNA near transcription sites (dot RNA). Further experiments showed that an exosome mutation coupled with transcriptional shutoff leads to a post-transcriptional increase in polyadenylated GAL1 3′ ends, which accompanies a loss of unadenylated (pA-) GAL1 RNA, a loss of post-transcriptional gene-periphery tethering as well as a decrease in dot RNA levels. This suggests that the exosome inhibits adenylation of some GAL1 transcripts, which results in the accumulation of pA-RNA adjacent to the GAL1 gene. We propose that this dot RNA, probably via RNP proteins, is an important component of the physical tether that links the GAL1 gene to the nuclear periphery. PMID:18614049

  16. Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms1

    PubMed Central

    Shannon, Jack C.; Pien, Fang-Mei; Cao, Heping; Liu, Kang-Chien

    1998-01-01

    Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels. PMID:9701580

  17. PPARγ-Dependent Regulation of Adenylate Cyclase 6 Amplifies the Stimulatory Effect of cAMP on Renin Gene Expression

    PubMed Central

    Desch, Michael; Schubert, Thomas; Schreiber, Andrea; Mayer, Sandra; Friedrich, Björn; Artunc, Ferruh; Todorov, Vladimir T.

    2010-01-01

    The second messenger cAMP plays an important role in the regulation of renin gene expression. Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) is known to stimulate renin gene transcription acting through PPARγ-binding sequences in renin promoter. We show now that activation of PPARγ by unsaturated fatty acids or thiazolidinediones drastically augments the cAMP-dependent increase of renin mRNA in the human renin-producing cell line Calu-6. The underlying mechanism involves potentiation of agonist-induced cAMP increase and up-regulation of adenylate cyclase 6 (AC6) gene expression. We identified a palindromic element with a 3-bp spacer (Pal3) in AC6 intron 1 (AC6Pal3). AC6Pal3 bound PPARγ and mediated trans-activation by PPARγ agonist. AC6 knockdown decreased basal renin mRNA level and attenuated the maximal PPARγ-dependent stimulation of the cAMP-induced renin gene expression. AC6Pal3 decoy oligonucleotide abrogated the PPARγ-dependent potentiation of cAMP-induced renin gene expression. Treatment of mice with PPARγ agonist increased AC6 mRNA kidney levels. Our data suggest that in addition to its direct effect on renin gene transcription, PPARγ “sensitizes” renin gene to cAMP via trans-activation of AC6 gene. AC6 has been identified as PPARγ target gene with a functional Pal3 sequence. PMID:20861226

  18. High expression of adenylate cyclase-associated protein 1 accelerates the proliferation, migration and invasion of neural glioma cells.

    PubMed

    Bao, Zhen; Qiu, Xiaojun; Wang, Donglin; Ban, Na; Fan, Shaochen; Chen, Wenjuan; Sun, Jie; Xing, Weikang; Wang, Yunfeng; Cui, Gang

    2016-04-01

    Adenylate cyclase-associated protein 1 (CAP1), a conserved member of cyclase-associated proteins was reported to be associated with the proliferation, migration or invasion of the tumors of pancreas, breast and liver, and was involved in astrocyte proliferation after acute Traumatic Brain Injury (TBI). In this study, we sought to investigate the character of CAP1 in the pathological process of human glioma by detecting human glioma specimens and cell lines. 43 of 100 specimens showed high expression of CAP1 via immunohistochemistry. With statistics analysis, we found out the expression level of CAP1 was correlated with the WHO grades of human glioma and was great positively related to Ki-67 (p<0.01). In vitro, silencing CAP1 in U251 and U87MG, the glioma cell lines with the relatively higher expression of CAP1, induced the proliferation of the cells significantly retarded, migration and invasion as well. Obviously, our results indicated that CAP1 participated in the molecular pathological process of glioma indeed, and in a certain sense, CAP1 might be a potential and promising molecular target for glioma diagnosis and therapies in the future.

  19. Behavioral effects of local microinfusion of pituitary adenylate cyclase activating polypeptide (PACAP) into the paraventricular nucleus of the hypothalamus (PVN).

    PubMed

    Norrholm, Seth D; Das, Mahasweta; Légrádi, Gábor

    2005-05-15

    Pituitary adenylate cyclase activating polypeptide (PACAP) has been implicated in the regulation of several autonomic and neuroendocrine functions. In the hypothalamic paraventricular nucleus (PVN), for example, PACAP-immunoreactive fibers densely innervate corticotropin-releasing hormone (CRH)-containing neurons in the medial parvocellular region, suggesting that PACAP acts to mediate stress responses. Therefore, we examined the behavioral effects of an intra-PVN PACAP injection (25 pmol) in combination with a mild stressor. PACAP or artificial cerebrospinal fluid (aCSF) was microinjected into the PVN (0.25 l) and then animals were restrained or placed in their home cage for 5 min. Exploratory activity (total distance traveled) and scored behaviors (face washing, body grooming, wet dog shakes, and rearing) were observed in a familiar open field for 10 min. In animals receiving aCSF, there were no behavioral differences between restrained and unrestrained groups. For the entire 10-min observation period, animals receiving PACAP, whether restrained or not, displayed elevated face washing and body grooming with decreased locomotor activity and rearing. Among PACAP-injected animals, restrained animals displayed increased body grooming compared to unrestrained animals during the first 2 min in the open field suggesting a summation of the effects of peptide injection and stressor. The observed elevation in grooming is consistent with previous studies reporting similar increases following electrical-, NMDA-, CRH-, or stressor-induced activation of the PVN. Thus, at the level of the PVN, PACAP may act as an excitatory neuropeptide and augment behavioral responses to stressors.

  20. The complete amino acid sequence of ubiquitin, an adenylate cyclase stimulating polypeptide probably universal in living cells.

    PubMed

    Schlesinger, D H; Goldstein, G; Niall, H D

    1975-05-20

    The complete amino acid sequence was determined for bovine ubiquitin, and adenylate cyclase stimulating polypeptide, which is probably represented universally in living cells. Ubiquitin has a molecular weight of 8451 and consists of a single polypeptide chain containing 74 amino acid residues. It contains four arginine residues but no cysteine or trytophan residues. The first 61 amino acid residues were obtained by automated Edman degradations. Tryptic digestion of maleated ubiquitin yielded four peptide fragments that were resolved by molecular sieve chromatography and coded in order of decreasing chain length (MT-1, MT-2, MT-3, and MT-4). The automated sequenator determinations on native ubiquintin provided overlapping sequence data for three of these fragments that gave an order of MT-1, MT-3, and then MT-2; Peptide MT-4, a dipeptide, was therefore assigned to the C terminus, and the placement of peptide MT-2 was corroborated by analysis of data from carboxypeptidase digestions of maleated ubiquitin. Peptide MT-2 was domaleated and sequenced by manual Edman degradations through a single lysine residue. It was cleaved at this residue with trypsin, and the two resultant peptides were separated by ion-exchange chromatography. Manual sequencing of the C-terminal demaleated tryptic peptide of MT-2 completed the sequence of MT-2 and that of native ubiquitin. The sequence of ubiquitin was further confirmed and supported by amino acid and parital sequence anlysis of fragments obtained by digestion of maleated ubiquitin with chymotrypsin or staphylococcal protease.

  1. Purification and primary structure of pituitary adenylate cyclase activating polypeptide (PACAP) from the brain of an elasmobranch, stingray, Dasyatis akajei.

    PubMed

    Matsuda, K; Yoshida, T; Nagano, Y; Kashimoto, K; Yatohgo, T; Shimomura, H; Shioda, S; Arimura, A; Uchiyama, M

    1998-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalami and found to exist as two amidated forms with 38 (PACAP 38) and 27 (PACAP 27) residues. The amino acid sequences of PACAPs isolated from the vertebrates, such as a bird, a frog and teleost fish, appear to be well conserved. In the present study, we attempted to isolate PACAP from the brain of an elasmobranch fish, Dasyatis akajei (stingray), which belongs to the Chondrichthyes (cartilaginous fish), by extraction of the acetone-dried powder with acetic acid, followed by successive high-performance liquid chromatography (HPLC) on a gel-filtration, a cation-exchange and two reverse-phase columns. Purification was monitored by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting analysis using an anti-PACAP 27 serum. The PACAP thus obtained consisted of 44 residues. The amino acid sequence of the comparable portion of its N-terminal 38 residues showed 92%, 89%, 89%, and 82% identity with those of mammalian, chicken, frog and teleost PACAPs with 38 residues, respectively. The extra six C-terminal residues of the stingray resembled those of tetrapod and teleost PACAP precursors which were deduced from the respective cDNAs. These results indicate that PACAP, which has an amino acid sequence showing high similarity with those of tetrapod and teleost PACAPs, is present in the elasmobranch brain.

  2. Content of N-6 methyl adenylic acid in heterogeneous nuclear and messenger RNA of HeLa cells.

    PubMed Central

    Lavi, U; Fernandez-Muñoz, R; Darnell, J E

    1977-01-01

    With the aid of a suitable thin layer chromatographic procedure, the N-6 methyl adenylic acid (m6A), content of a variety of 32P labeled RNA species from HeLa cells has been measured. Poly(A)-containing (poly(A)+) cytoplasmic RNA has on the average one m6Ap per 800 to 900 nucleotides. This value is independent of the length of the molecules. The proportion of m6Ap in poly(A)+ cytoplasmic RNA does not change between 4 and 18 hours of labeling with 32P, suggesting that the majority of the messenger RNA molecules may have a similar level of internal methylation regardless of their half-life. The non-polyadenylated, non-ribosomal cytoplasmic RNA fraction sedimenting from 10S TO 28S is less methylated with approximately one m6A per 2,700 nucleotides. Heterogeneous nuclear RNA molecules (DMSO treated) which sediment from 28S to 45S have approximately one m6Ap per 3,000 nucleotides. The hnRNA molecules sedimenting from 10S to 28S have one m6Ap per 1,800 nucleotides. Poly(A)+ nuclear RNA is enriched in m6A, containing 1 residue of m6A per 700 to 800 nucleotides, a value close to that obtained for the polyadenylated cytoplasmic RNA. Images PMID:866178

  3. Oxidative Stress Tolerance, Adenylate Cyclase, and Autophagy Are Key Players in the Chronological Life Span of Saccharomyces cerevisiae during Winemaking

    PubMed Central

    Orozco, Helena; Matallana, Emilia

    2012-01-01

    Most grape juice fermentation takes place when yeast cells are in a nondividing state called the stationary phase. Under such circumstances, we aimed to identify the genetic determinants controlling longevity, known as the chronological life span. We identified commercial strains with both short (EC1118) and long (CSM) life spans in laboratory growth medium and compared them under diverse conditions. Strain CSM shows better tolerance to stresses, including oxidative stress, in the stationary phase. This is reflected during winemaking, when this strain has an increased maximum life span. Compared to EC1118, CSM overexpresses a mitochondrial rhodanese gene-like gene, RDL2, whose deletion leads to increased reactive oxygen species production at the end of fermentation and a correlative loss of viability at this point. EC1118 shows faster growth and higher expression of glycolytic genes, and this is related to greater PKA activity due to the upregulation of the adenylate cyclase gene. This phenotype has been linked to the presence of a δ element in its promoter, whose removal increases the life span. Finally, EC1118 exhibits a higher level of protein degradation by autophagy, which might help achieve fast growth at the expense of cellular structures and may be relevant for long-term survival under winemaking conditions. PMID:22327582

  4. Molecular cloning, subcellular localization and characterization of two adenylate kinases from cassava, Manihot esculenta Crantz cv. KU50.

    PubMed

    Boonrueng, Channarong; Tangpranomkorn, Surachat; Yazhisai, Uthaman; Sirikantaramas, Supaart

    2016-10-01

    Adenylate kinase (ADK) is a phosphotransferase that plays an important role in cellular energy homeostasis. Many isozymes located in different subcellular compartments have been reported. In this study, we focus on the characterization of cassava (Manihot esculenta) ADKs. We found 15 ADKs that are publicly available in the African cassava genome database. We cloned two ADKs, namely MeADK1 and MeADK2, which are phylogenetically grouped together with the plastidial ADK in potato. Both MeADK1 and MeADK2 showed 66% identity in the amino acid sequences with plastidial ADK in potato. However, we demonstrated that they are localized to mitochondria using GFP fusions of MeADK1 and MeADK2. The Escherichia coli-produced recombinant MeADK1 and MeADK2 preferred forward reactions that produce ATP. They exhibited similar specific activities. The semi-quantitative RT-PCR analysis showed that MeADK1 and MeADK2 in 2-month-old leaves have similar expression patterns under a diurnal light-dark cycle. However, MeADK2 transcripts were expressed at much higher levels than MeADK1 in 5-month-old leaves and roots. Thus, we conclude that MeADK2 might play a vital role in energy homeostasis in cassava mitochondria.

  5. Role of Bordetella pertussis RseA in the cell envelope stress response and adenylate cyclase toxin release

    PubMed Central

    Hanawa, Tomoko; Yonezawa, Hideo; Kawakami, Hayato; Kamiya, Shigeru; Armstrong, Sandra K.

    2013-01-01

    Bordetella pertussis is the bacterial agent of the human disease, whooping cough. In many bacteria, the extracellular function sigma factor σE is central to the response to envelope stress, and its activity is negatively controlled by the RseA anti-sigma factor. In this study, the role of RseA in B. pertussis envelope stress responses was investigated. Compared with the wild-type strain, an rseA mutant showed elevated resistance to envelope stress and enhanced growth at 25°C. rpoH and other predicted σE target genes demonstrated increased transcription in the rseA mutant compared with the wild type parent. Transcription of those genes was also increased in wild type B. pertussis and Escherichia coli under envelope stress, whereas no stress-induced increase in transcription was observed in the rseA mutant. rseA inactivation was also associated with altered levels of certain proteins in culture supernatant fluids, which showed increased adenylate cyclase toxin (CyaA) levels. The increased CyaA in the mutant was correlated with an apparent increased stability of the extracellular toxin and increased production of CyaA-containing outer membrane vesicles. Consistent with this, compared with the wild type strain, rseA mutant cells produced increased numbers of large surface-associated vesicles. PMID:23821542

  6. Stress tolerance of the Saccharomyces cerevisiae adenylate cyclase fil1 (CYR1) mutant depends on Hsp26.

    PubMed

    Vianna, Cristina R; Ferreira, Mariana C; Silva, Carol L C; Tanghe, An; Neves, Maria J; Thevelein, Johan M; Rosa, Carlos A; Van Dijck, Patrick

    2010-01-01

    Fermentation-induced loss of stress resistance in yeast is an important phenotype from an industrial point of view. It hampers optimal use of frozen dough applications as well as high gravity brewing fermentations because these applications require stress-tolerant yeast strains during active fermentation. Different mutants (e.g. fil1, an adenylate cyclase mutant CYR1(lys1682)) that are affected in this loss of stress resistance have been isolated, but so far the identification of the target genes important for the increased tolerance has failed. Previously we have shown that neither trehalose nor Hsp104 nor STRE-controlled genes are involved in the higher stress tolerance of the fil1 mutant. The contribution of other putative downstream factors of the PKA pathway was investigated and here we show that the small heat-shock protein Hsp26 is required for the high heat stress tolerance of the fil1 mutant, both in stationary phase cells as well as during active fermentation.

  7. Insolubilization process increases enzyme stability

    NASA Technical Reports Server (NTRS)

    Billingham, J.; Lyn, J.

    1971-01-01

    Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes.

  8. The relationship between the occupation of the D-1 dopamine receptor by [3H]piflutixol and the activity of dopamine-sensitive adenylate cyclase in rat striatal membranes.

    PubMed

    Fleminger, S

    1991-07-05

    The relationship between occupation of the D-1 dopamine receptor by [3H]piflutixol and inhibition of dopamine-sensitive adenylate cyclase has been studied. Experiments were performed in parallel; after the initial incubation to enable binding of [3H]piflutixol, half the tubes were assayed for [3H]piflutixol binding and the other half assayed for adenylate cyclase activity. The assay conditions for the two halves of the experiments were identical. (+/-)Sulpiride (3 x 10(-5)M) was present in all tubes to mask drug binding to the D-2 receptor. The inhibition of dopamine- (10(-3) and 10(-5)M) sensitive adenylate cyclase with increasing concentrations of [3H]piflutixol in the incubation mixture was compared to the saturation of specific [3H]piflutixol binding with those same concentrations of [3H]piflutixol. There was a linear relationship between receptor occupation by [3H]piflutixol and inhibition of dopamine sensitive adenylate cyclase. In a second experiment dopamine was present during the initial incubation with [3H]piflutixol. This resulted in a displacement of specific [3H]piflutixol binding and, as a consequence, a reduction of [3H]piflutixol's inhibition of dopamine-sensitive adenylate cyclase. In the absence of GTP in the initial incubation dopamine produced a greater reduction of [3H]piflutixol's inhibition of dopamine adenylate cyclase than displacement of specific [3H]piflutixol binding. In the presence of GTP in the initial incubation both displacement curves were shifted to the right, i.e. dopamine was less potent. However, under these conditions dopamine produced less inhibition of [3H]piflutixol's inhibition of dopamine adenylate cyclase than displacement of specific [3H]piflutixol binding. These results are interpreted as resulting from changes in D-1high and D-1low ratios as a result of incubation in the presence or absence of GTP.

  9. Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL) with Nucleoside-Based Bisubstrate Adenylation Inhibitors

    PubMed Central

    Petrelli, Riccardo; De la Mora-Rey, Teresa; Tiwari, Divya; Liu, Feng; Dawadi, Surrendra; Nandakumar, Madhumitha; Rhee, Kyu Y.; Schnappinger, Dirk; Finzel, Barry C.; Aldrich, Courtney C.

    2015-01-01

    Mycobacterium tuberculosis (Mtb) responsible for both latent and symptomatic tuberculosis (TB) remains the second leading cause of mortality among infectious diseases worldwide. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. We report the synthesis and evaluation of a systematic series of potent nucleoside-based inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with KD's below 2 nM. Additionally, we obtained high-resolution co-crystal structures for a majority of the compounds. Despite fairly uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MIC) ranging from 0.78 to >100 μM. Cellular accumulation studies showed a nearly 10-fold enhanced accumulation of a C-2′-α analog over the corresponding C-2′-β analog, consistent with their differential whole-cell activity. PMID:26299766

  10. Developments in Enzyme Technology.

    ERIC Educational Resources Information Center

    Chaplin, M. F.

    1984-01-01

    Enzyme technology has a well-established industrial base, with applications that have survived competition. The most prominent applications of enzymes in biotechnology are examined with an explanation of some theoretical background. Topics include extending an enzyme's useful life, partition and diffusion, industrial uses, and therapeutic uses.…

  11. Developments in Enzyme Technology.

    ERIC Educational Resources Information Center

    Chaplin, M. F.

    1984-01-01

    Enzyme technology has a well-established industrial base, with applications that have survived competition. The most prominent applications of enzymes in biotechnology are examined with an explanation of some theoretical background. Topics include extending an enzyme's useful life, partition and diffusion, industrial uses, and therapeutic uses.…

  12. Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties.

    PubMed

    Kuby, S A; Hamada, M; Johnson, M S; Russell, G A; Manship, M; Palmieri, R H; Fleming, G; Bredt, D S; Mildvan, A S

    1989-08-01

    Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamada et al., 1979). By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP2- and MgADP-) and (b) the uncomplexed nucleotide substrates (ADP3- and AMP2-) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15-25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of epsilon AMP. The syntheses are described as a set of peptides corresponding to residues 31-45, 20-45, 5-45, and 1-45, and a set of peptides corresponding to residues 178-192, 178-194, and 172-194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: epsilon MgATP/epsilon ATP and epsilon MgADP/epsilon ADP are quantitatively presented in terms of their intrinsic dissociation constants (K'd) and values of N (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1-44) obtained from rabbit muscle myokinase (Kuby et al., 1984) and with the native enzyme (Hamada et al., 1979). In addition, the values of N and K'd are given for the second set of synthetic peptides to the fluorescent ligands epsilon AMP and epsilon ADP as well