The Experts below are selected from a list of 228 Experts worldwide ranked by ideXlab platform
Roland Seifert - One of the best experts on this subject based on the ideXlab platform.
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nucleotidyl cyclase activity of particulate guanylyl cyclase a comparison with particulate guanylyl cyclases e and f soluble guanylyl cyclase and bacterial adenylyl cyclases cyaa and edema factor
PLOS ONE, 2013Co-Authors: Kerstin Y Beste, Corinna Spangler, Karlwilhelm Koch, Weijen Tang, Yuequan Shen, Volkhard Kaever, Heike Burhenne, Roland SeifertAbstract:Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.
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structural basis for the high affinity inhibition of mammalian membranous adenylyl cyclase by 2 3 o n methylanthraniloyl inosine 5 triphosphate
Molecular Pharmacology, 2011Co-Authors: Melanie Hubner, Anshuman Dixit, Tungchung Mou, Gerald H Lushington, Cibele S Pinto, Andreas Gille, Jens Geduhn, Stephen R Sprang, Burkhard Konig, Roland SeifertAbstract:2',3'-O-(N-Methylanthraniloyl)-inosine 5'-triphosphate (MANT-ITP) is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, Ki, 1 nM) yet discovered and surpasses the potency of 2',3'-O-(N-methylanthraniloyl)-guanosine 5'-triphosphate (MANT-GTP) 55-fold (Gottle et al., J Pharmacol Exp Ther 329:1156-1165 (2009)). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP. MANT-ITP was a considerably more potent inhibitor of the purified catalytic domains VC1 and IIC2 of mAC than MANT-GTP (Ki, 0.7 nM versus 18 nM). Moreover, there was considerably more efficient fluorescence resonance energy transfer between W1020 of IIC2 and the MANT-group of MANT-ITP compared to MANT-GTP, indicating optimal interaction of the MANT-group of MANT-ITP with the hydrophobic pocket. The crystal structure of MANT-ITP in complex with the Gsalpha- and forskolin-activated catalytic domains VC1:IIC2 in comparison to the existing MANT-GTP crystal structure revealed only subtle differences in binding mode. The higher affinity of MANT-ITP to mAC compared to MANT-GTP is probably due to fewer stereochemical constraints upon the nucleotide base in the purine binding pocket, allowing a stronger interaction with the hydrophobic regions of IIC2 domain, as assessed by fluorescence spectroscopy. Stronger interaction is also achieved in the phosphate-binding site. The triphosphate group of MANT-ITP exhibits better metal coordination than the triphosphate group of MANT-GTP, as confirmed by molecular dynamics simulations. Collectively, the subtle differences in ligand structure have profound effects on affinity for mAC.
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structural basis for the high affinity inhibition of mammalian membranous adenylyl cyclase by 2 3 o n methylanthraniloyl inosine 5 triphosphate s
2011Co-Authors: Anshuman Dixit, Tungchung Mou, Gerald H Lushington, Cibele S Pinto, Andreas Gille, Jens Geduhn, Stephen R Sprang, Roland SeifertAbstract:O-(N-Methylanthraniloyl)-ITP (MANT-ITP) is the most po- tent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, Ki, 1 nM) yet discovered and surpasses the potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329: 1156-1165, 2009). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP. MANT-ITP was a considerably more potent inhibitor of the purified catalytic domains VC1 and IIC2 of mAC than MANT-GTP (Ki, 0.7 versus 18 nM). Moreover, there was con- siderably more efficient fluorescence resonance energy transfer between Trp1020 of IIC2 and the MANT group of MANT-ITP compared with MANT-GTP, indicating optimal interaction of the MANT group of MANT-ITP with the hydrophobic pocket. The crystal structure of MANT-ITP in complex with the Gs- and forskolin-activated catalytic domains VC1:IIC2 compared with the existing MANT-GTP crystal structure revealed only subtle differences in binding mode. The higher affinity of MANT-ITP to mAC compared with MANT-GTP is probably due to fewer stereochemical constraints upon the nucleotide base in the purine binding pocket, allowing a stronger interaction with the hydrophobic regions of IIC2 domain, as assessed by fluorescence spectroscopy. Stronger interaction is also achieved in the phosphate-binding site. The triphosphate group of MANT-ITP exhibits better metal coordination than the triphosphate group of MANT-GTP, as confirmed by molecular dynamics simulations. Collectively, the subtle differences in ligand structure have profound effects on affinity for mAC.
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functionally nonequivalent interactions of guanosine 5 triphosphate inosine 5 triphosphate and xanthosine 5 triphosphate with the retinal g protein transducin and with gi proteins in hl 60 leukemia cell membranes
Biochemical Pharmacology, 1997Co-Authors: Jan F Klinker, Roland SeifertAbstract:Abstract G-proteins mediate signal transfer from receptors to effector systems. In their guanosine 5′-triphosphate (GTP)-bound form, G-protein α-subunits activate effector systems. Termination of G-protein activation is achieved by the high-affinity GTPase [E.C. 3.6.1.] of their α-subunits. Like GTP, inosine 5′-triphosphate (ITP) and xanthosine 5′-triphosphate (XTP) can support effector system activation. We studied the interactions of GTP, ITP, and XTP with the retinal G-protein, transducin (TD), and with G-proteins in HL-60 leukemia cell membranes. TD hydrolyzed nucleoside 5′-triphosphates (NTPs) in the order of efficacy GTP > ITP > XTP. NTPs eluted TD from rod outer segment disk membranes in the same order of efficacy. ITP and XTP competitively inhibited TD-catalyzed GTP hydrolysis. In HL-60 membranes, the chemoattractants N-formyl- l -methionyl- l -leucyl- l -phenylalanine (fMLP) and leukotriene B 4 (LTB 4 ) effectively activated GTP and ITP hydrolysis by G i -proteins. fMLP and LTB 4 were at least 10-fold more potent activators of ITPase than of GTPase. Complement C5a effectively activated the GTPase of G i -proteins but was only a weak stimulator of ITPase. The potency of C5a to activate GTP and ITP hydrolysis was similar. The fMLP-stimulated GTPase had a lower K m value than the fMLP-stimulated ITPase, whereas the opposite was true for the V max values. fMLP, C5a, and LTB 4 did not stimulate XTP hydrolysis. Collectively, our data show that GTP, ITP, and XTP bind to G-proteins with different affinities, that G-proteins hydrolyze NTPs with different efficacies, and that chemoattractants stimulate GTP and ITP hydrolysis by G i -proteins in a receptor-specific manner. On the basis of our results and the data in the literature, we put forward the hypothesis that GTP, ITP, and XTP act as differential signal amplifiers and signal sorters at the G-protein level.
G.p. Connolly - One of the best experts on this subject based on the ideXlab platform.
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structure activity relationship of a pyrimidine receptor in the rat isolated superior cervical ganglion
British Journal of Pharmacology, 1995Co-Authors: G.p. Connolly, Paul J. HarrisonAbstract:Abstract 1. The effects of pyrimidines and purines on the d.c. potential of the rat isolated superior cervical ganglion (SCG) have been examined by a grease-gap technique to determine the structure-activity requirements of the receptor activated by pyrimidines, i.e. a pyrimidinoceptor. 2. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl (ZTP), the pyrimidines, cytidine 5'-triphosphate (CTP), uridine 5'-triphosphate (UTP) and thymidine 5'-triphosphate (TTP) and the purines, adenosine 5'-triphosphate (ATP; in the presence of an A1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 microM)), adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), guanosine 5'-triphosphate (GTP), inosine 5'-triphosphate (1TP) depolarized ganglia in a concentration-dependent manner. The relative order of ZTP and purine 5'-triphosphates in depolarizing ganglia was ZTP > or = ATP gamma S > > ATP > or = ITP = GTP, and for the pyrimidine 5'-triphosphates UTP > TTP > or = CTP. Depolarizations evoked by ATP gamma S were followed by concentration-dependent hyperpolarizations at 100 and 1000 microM. 3. At concentrations of between 0.1 microM and 1 mM, uridine 5'-diphosphate (UDP), uridine 5'-diphosphoglucose (UDPG) and uridine 5'-diphosphoglucuronic acid (UDPGA) evoked significant and concentration-dependent depolarizations, whereas uridine 5'-monophosphate (UMP), uridine and uracil were inactive or produced small ( or = UTP > UDPG > UDPGA > > uracil > or = UMP = pseudouridine > or = uridine. At 3 and 10 mM, uridine produced concentration-dependent hyperpolarizations. Nikkomycin Z, a nucleoside resembling UTP (viz. the triphosphate chain at the 5'-position on the ribose moiety being replaced by a peptide), was inactive between 1 microM and 1 mM. Generally, a concentration of 10 mM was required before thymidine, 6-azathymine, 6-azauracil or 6-azauridine depolarized ganglia. 4. Suramin (300 microM), a P2-purinoceptor antagonist, significantly depressed depolarizations evoked by alpha, beta-methylene-ATP (alpha, beta-MeATP; 100 microM), ATP gamma S (100 microM), CTP (1 mM), GTP (1 mM), ZTP (30 microM) and ATP (300 microM) in the presence of DPCPX (1 microM). Suramin reversed a small depolarization evoked by UMP (1 mM) into a small hyperpolarization. In contrast depolarizations evoked by UDP, UTP, UDPG (all at 100 microM) and TTP (300 microM) were unaltered or enhanced by suramin. 5. It is concluded that the rat SCG contains distinct nucleotide receptors including a P2-purinoceptor (activated by alpha, beta-MeATP, ATP, GTP, ITP and ZTP) and a pyrimidinoceptor (activated by UTP, UDP, UDPG, UDPGA and TTP). The pyrimidinoceptor on rat SCG neurones had specific structure activity requirements with the di- and triphosphates of uridine being the most effective depolarizing agonists examined.
Paul J. Harrison - One of the best experts on this subject based on the ideXlab platform.
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structure activity relationship of a pyrimidine receptor in the rat isolated superior cervical ganglion
British Journal of Pharmacology, 1995Co-Authors: G.p. Connolly, Paul J. HarrisonAbstract:Abstract 1. The effects of pyrimidines and purines on the d.c. potential of the rat isolated superior cervical ganglion (SCG) have been examined by a grease-gap technique to determine the structure-activity requirements of the receptor activated by pyrimidines, i.e. a pyrimidinoceptor. 2. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl (ZTP), the pyrimidines, cytidine 5'-triphosphate (CTP), uridine 5'-triphosphate (UTP) and thymidine 5'-triphosphate (TTP) and the purines, adenosine 5'-triphosphate (ATP; in the presence of an A1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 microM)), adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), guanosine 5'-triphosphate (GTP), inosine 5'-triphosphate (1TP) depolarized ganglia in a concentration-dependent manner. The relative order of ZTP and purine 5'-triphosphates in depolarizing ganglia was ZTP > or = ATP gamma S > > ATP > or = ITP = GTP, and for the pyrimidine 5'-triphosphates UTP > TTP > or = CTP. Depolarizations evoked by ATP gamma S were followed by concentration-dependent hyperpolarizations at 100 and 1000 microM. 3. At concentrations of between 0.1 microM and 1 mM, uridine 5'-diphosphate (UDP), uridine 5'-diphosphoglucose (UDPG) and uridine 5'-diphosphoglucuronic acid (UDPGA) evoked significant and concentration-dependent depolarizations, whereas uridine 5'-monophosphate (UMP), uridine and uracil were inactive or produced small ( or = UTP > UDPG > UDPGA > > uracil > or = UMP = pseudouridine > or = uridine. At 3 and 10 mM, uridine produced concentration-dependent hyperpolarizations. Nikkomycin Z, a nucleoside resembling UTP (viz. the triphosphate chain at the 5'-position on the ribose moiety being replaced by a peptide), was inactive between 1 microM and 1 mM. Generally, a concentration of 10 mM was required before thymidine, 6-azathymine, 6-azauracil or 6-azauridine depolarized ganglia. 4. Suramin (300 microM), a P2-purinoceptor antagonist, significantly depressed depolarizations evoked by alpha, beta-methylene-ATP (alpha, beta-MeATP; 100 microM), ATP gamma S (100 microM), CTP (1 mM), GTP (1 mM), ZTP (30 microM) and ATP (300 microM) in the presence of DPCPX (1 microM). Suramin reversed a small depolarization evoked by UMP (1 mM) into a small hyperpolarization. In contrast depolarizations evoked by UDP, UTP, UDPG (all at 100 microM) and TTP (300 microM) were unaltered or enhanced by suramin. 5. It is concluded that the rat SCG contains distinct nucleotide receptors including a P2-purinoceptor (activated by alpha, beta-MeATP, ATP, GTP, ITP and ZTP) and a pyrimidinoceptor (activated by UTP, UDP, UDPG, UDPGA and TTP). The pyrimidinoceptor on rat SCG neurones had specific structure activity requirements with the di- and triphosphates of uridine being the most effective depolarizing agonists examined.
Helmut Sigel - One of the best experts on this subject based on the ideXlab platform.
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stabilities and isomeric equilibria in solutions of monomeric metal ion complexes of guanosine 5 triphosphate gtp4 and inosine 5 triphosphate ITP4 in comparison with those of adenosine 5 triphosphate atp4
Chemistry: A European Journal, 2001Co-Authors: Helmut Sigel, Nicolas A Corfu, Emanuela M Bianchi, Yoshiaki Kinjo, Roger Tribolet, Bruce R MartinAbstract:Under experimental conditions in which the self-association of the purine-nucleoside 5′-triphosphates (PuNTPs) GTP and ITP is negligible, potentiometric pH titrations were carried out to determine the stabilities of the M(H;PuNTP)− and M(PuNTP)2− complexes where M2+=Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, or Cd2+ (I=0.1 M, 25 °C). The stabilities of all M(GTP)2− and M(ITP)2− complexes are significantly larger than those of the corresponding complexes formed with pyrimidine-nucleoside 5′-triphosphates (PyNTPs), which had been determined previously under the same conditions. This increased complex stability is attributed, in agreement with previous 1H MNR shift studies, to the formation of macrochelates of the phosphate-coordinated metal ions with N7 of the purine residues. A similar enhanced stability (despite relatively large error limits) was observed for the M(H;PuNTP)− complexes, in which H+ is bound to the terminal γ-phosphate group, relative to the stability of the M(H;PyNTP)− species. The percentage of the macrochelated isomers in the M(GTP)2− and M(ITP)2− systems was quantified by employing the difference log K−log K; the lowest and highest formation degrees of the macrochelates were observed for Mg(ITP)2− and Cu(GTP)2− with 17±11 % and 97±1 %, respectively. From previous studies of M(ATP)2− complexes, it is known that innersphere and outersphere macrochelates may form; that is, in the latter case a water molecule is between N7 and the phosphate-coordinated M2+. Similar conclusions are reached now by comparisons with earlier 1H MNR shift measurements, that is, that Mg(GTP)2− (21±11 %), for example, exists largely in the form of an outersphere macrochelate and Zn(GTP)2− (68±4 %) as an innersphere one. Generally, the overall percentage of macrochelate falls off for a given metal ion in the order M(GTP)2−>M(ITP)2−>M(ATP)2−; this is in accord with the decreasing basicity of N7 and the steric inhibition of the (C6)NH2 group in the adenine residue. Furthermore, although the absolute stability constants of the previously studied M(GMP), M(IMP), and M(AMP) complexes differ by about two to three log units from the present M(PuNTP)2− results, the formation degrees of the macrochelates are astonishingly similar for the two series of nucleotides for a given metal ion and purine-nucleobase residue. The conclusion that N7 of the guanine residue is an especially favored binding site for metal ions is also in accord with observations made for nucleic acids.
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acid base properties of nucleosides and nucleotides as a function of concentration comparison of the proton affinity of the nucleic base residues in the monomeric and self associated oligomeric 5 triphosphates of inosine ITP guanosine gtp and adenosi
FEBS Journal, 1991Co-Authors: Nicolas A Corfu, Helmut SigelAbstract:The acid-base properties of the nucleic base residues of ITP, GTP, and ATP, and for comparison also as far as possible of the corresponding nucleosides, were studied in dependence on their concentration, i. e. on the effect of self-association. From the dependence between the 1H-NMR chemical shifts of H-2 (where applicable), H-8, and H-1′, and the pD of the solutions, the acidity constants for the deprotonation of the D+ (N-7) site in D2(ITP)2−, D2(GTP)2−, D(Ino)+, and D(Guo)+, and of the D+ (N-1) site in D2(ATP)2− and D(Ado)+ were calculated. Chemical shift/pD profiles for a whole series of varying concentrations of the nucleic base derivatives (= N) were constructed, including those for infinite dilution (δo), which give the acidity constant for the monomeric N, and for infinitely concentrated solutions (δ∞), which give the acidity constant of an N in an infinitely long stack. The acidity constants determined from the δo/pD plots are in excellent agreement with the pKa values measured by potentiometric pH titrations of highly diluted solutions of N. The effects of self-association are striking: e.g. the pKa value of the D+ (N-7) site in D2(GTP)2− is lowered by about 1 (as calculated from the δo/pD and δ∞/pD profiles), while the pKa value of the D+ (N-1) site in D2(ATP)2− is increased by approximately 0.3; i.e. in the first case deprotonation is facilitated and in the second it is inhibited. The increasing inhibition of the H+ (N-1) deprotonation with an increasing ATP concentration is due to the high stability of the dimeric [H2(ATP)]24− stack for which the intermolecular H+(N-1)/γ-P(OH)(O)2− ion pairs between the two ATP molecules are crucial. In those cases where no other significant interaction but aromatic-ring stacking in the self-association process occurs, the release of protons from protonated nitrogen-ring sites is facilitated with increasing stacking; this holds not only for D2(GTP)2− as indicated above, but also for D2(ITP)2−, D(Ino)+, and D(Ado)+. The latter example especially suggests that the situation for the D2(ATP)2− system is exceptional. Some consequences of the considered acid-base properties for biological sytems are indicated.
Michael J Welsh - One of the best experts on this subject based on the ideXlab platform.
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nucleoside triphosphates are required to open the cftr chloride channel
Cell, 1991Co-Authors: Matthew P Anderson, Herbert A Berger, Devra P Rich, Richard J Gregory, Alan E Smith, Michael J WelshAbstract:Abstract The CFTR Cl − channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATPγS substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP > GTP > ITP ≈ UTP > CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg 2+ -free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF-associated mutations in the NBDs block Cl − channel function.
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nucleoside triphosphates are required to open the cftr chloride channel
Cell, 1991Co-Authors: Matthew P Anderson, Herbert A Berger, Devra P Rich, Richard J Gregory, Alan E Smith, Michael J WelshAbstract:The CFTR Cl- channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATP gamma S substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP greater than GTP greater than ITP approximately UTP greater than CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg(2+)-free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF-associated mutations in the NBDs block Cl- channel function.
