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Query: UNIPROT:P06889 (Mol)
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We investigated the roles of cyclic GMP and cyclic AMP in the inhibition of rabbit platelet aggregation and degranulation by two nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1; the active metabolite of molsidomine), with particular reference to the synergistic interaction of these drugs with prostaglandin E1 (PGE1). Changes in platelet cyclic [3H]GMP and cyclic [3H]AMP were measured by rapid and sensitive prelabeling techniques, the validity of which were confirmed by radioimmunoassays. Incubation of the platelets with 0.1 to 10 microM SNP alone for 0.5 min caused progressively greater inhibitions of platelet function associated with large dose-dependent increases in cyclic [3H]GMP and 1.4- to 3.0-fold increases in cyclic [3H]AMP. However, addition of SNP with the adenylate cyclase activator, PGE1, at a concentration of the latter that had little effect alone, caused much larger increases in cyclic [3H]AMP and greatly enhanced the inhibition of platelet aggregation. SIN-1 had effects similar to those of SNP, although it was less active. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) diminished the increases in cyclic [3H]AMP caused by SNP or SIN-1 in both the presence and absence of PGE1 but reduced the inhibition of platelet function caused by the nitrovasodilators only in the presence of PGE1. These results suggest that, although cyclic GMP may mediate the inhibition of rabbit platelet function by high concentrations of nitrovasodilators added alone, the synergistic interaction of lower concentrations with PGE1 depends on an enhanced accumulation of cyclic AMP. Synergistic effects on cyclic [3H]AMP accumulation were also observed on incubation of platelets with SNP and adenosine, another activator of adenylate cyclase. Hemoglobin, which binds nitric oxide, blocked or reversed the increases in both cyclic [3H]GMP and cyclic [3H]AMP in platelets caused by the nitrovasodilators added either alone or with PGE1. Cilostamide, a selective inhibitor of platelet low Km cyclic AMP phosphodiesterase, had effects on platelet cyclic [3H]AMP accumulation identical to those of SNP, suggesting that the action of the latter depends on inhibition of the same enzyme. M&B 22,948, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in both cyclic [3H]GMP and cyclic [3H]AMP caused by SNP. A hyperbolic relationship was found between the increases in cyclic [3H]GMP and cyclic [3H]AMP caused by different concentrations of SNP; this relationship was not affected by addition of M&B 22,948. The results strongly suggest that the increases in platelet cyclic [3H]AMP caused by nitrovasodilators in the presence or absence of activators of adenylate cyclase are mediated by the inhibition by cyclic GMP of cyclic AMP breakdown.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1990 May
PMID:Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: inhibition of cyclic AMP breakdown by cyclic GMP. 216 60

A computer simulation (KINSIM) modeling up to 33 competing reactions was used in order to investigate the product distribution in a template-directed oligonucleotide synthesis as a function of time and concentration of the reactants. The study is focused on the poly(C)-directed elongation reaction of an oligoguanylate (a 7-mer is chosen) with guanosine 5'-monophosphate-2-methyl-imidazolide (2-MeImpG), the activated monomer. It is known that the elongation of oligoguanylates to form oligomeric products such as 8-mer, 9-mer, 10-mer, etc., is in competition with (1) the dimerization and further oligomerization reaction of 2-MeImpG that leads to the formation of dimers and short oligomers, and (2) the hydrolysis of 2-MeImpG that forms inactive guanosine 5'-monophosphate, 5'-GMP. Experimentally determined rate constants for the above three processes at 37 degrees C and pH 7.95 were used in the simulation; the initial concentrations of 2-MeImpG, [M]o, and of the oligoguanylate primer, [7-mer]o, were varied, and KINSIM calculated the distribution of products as a function of time until equilibration was reached, i.e., when all the activated monomer has been consumed. In order to sort out how strongly the elongation reaction may be affected by the competing hydrolysis and dimerization, we also simulated the idealized situation in which these competing reactions do not occur.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Evol 1990 Dec
PMID:Computer simulation in template-directed oligonucleotide synthesis. 217 95

The possibility, that a GTP-binding protein is involved in the transducing mechanism leading to the formation of inositol trisphosphate (InsP3) in heart was explored in rat heart ventricles. Accordingly, a crude membrane fraction was isolated from 3[H] inositol prelabelled rat heart ventricles. When incubated with the non-hydrolysable GTP analogues GTP gamma S and GMP-PNP, it produced InsP3 in a time- and concentration-dependent manner. GDP beta S and the aminoglycoside antibiotic neomycin were effective inhibitors of this activation. In the absence of GTP gamma S or GMP-PNP, no such formation occurred with Ca2+ concentration from 10 nM to 1 microM but formation tripled in relation to the control level when Ca2+ concentration was raised from 1 microM to 100 microM. GTP gamma S increased the Ca2+ sensitivity of InsP3 production towards more physiologically relevant concentrations occurring during diastole (100 nM). These findings strongly suggest the presence in heart of a particulate Ca2(+)-dependent phospholipase C, whose activity is regulated by guanine nucleotides. This Ca2(+)-dependent phospholipase C observed in a cell free system was evidenced also in a multicellular system when altering the free Ca2+ concentrations around the physiological range. The results support the possibility that the enzyme might be activated during each cardiac cycle and thus produce two potential activators of cardiac contraction, namely InsP3 and diglycerides.
J Mol Cell Cardiol 1990 Jan
PMID:Mediation by GTP gamma S and Ca2+ of inositol trisphosphate generation in rat heart membranes. 218 85

A 32P-labelled ATP analog, 3'-O-(4-benzoyl)benzoyl ATP (BzATP) previously shown to be an agonist at P2Y-purinergic receptors (Boyer J. L., and Harden T. K. (1989) Mol. Pharmacol. 36, 831-835), has been used as a probe for the P2Y-purinergic receptor on turkey erythrocyte plasma membranes. In the absence of light, [32P]BzATP bound to membranes with high affinity (KD approximately 5 nM), and in a saturable and reversible manner. The binding of [32P]BzATP was competitively inhibited by ATP and ADP analogs (2-methylthioadenosine 5'-triphosphate greater than adenosine 5'-O-(2-thiodiphosphate) greater than BzATP greater than ATP greater than beta,gamma-methyleneadenosine 5'-triphosphate greater than 5'-adenylylimidodiphosphate) with pharmacological specificity consistent with that of a P2Y-purinergic receptor. Guanine nucleotides (guanosine 5'-O-(3-thiotriphosphate) greater than GTP greater than guanosine 5'-O-(2-thiodiphosphate) greater than GMP) noncompetitively inhibited the binding of radioligand. Photolysis of [32P] BzATP-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 53,000 Da. Photolabeling was inhibited in a concentration-dependent manner by ATP and ADP analogs with a potency order characteristic for a P2Y-purinergic receptor and was modulated by guanine nucleotides. A protein of approximately 53,000 daltons was also labeled by [32P]BzATP in membranes from several other tissues known to express the P2Y-purinergic receptor. These results suggest that [32P]BzATP can be used to label covalently the P2Y-purinergic receptor and that this radioprobe will be a useful reagent for further characterization and purification of the P2Y-purinergic receptor.
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PMID:[32P]3'-O-(4-benzoyl)benzoyl ATP as a photoaffinity label for a phospholipase C-coupled P2Y-purinergic receptor. 219 38

The base-pairing fidelity of DNA primase from calf thymus was studied in vitro by using a misinsertion assay based on synthetic polydeoxynucleotide templates. With poly(dT) as template, GMP misinsertions occurred with a frequency of one error per 1600 correctly incorporated nucleotides, while UMP and CMP were inserted with a frequency of 1/300 and 1/500, respectively. Accuracy with poly(dC, dT) as template was 1/200 for the misinsertion of UMP, and 1/300 for the misinsertion of CMP. Thus, DNA primase is the least accurate polynucleotide-synthesizing enzyme known. The results are discussed in terms of an obvious necessity for a priming mechanism at the beginning of DNA synthesis.
J Mol Biol 1990 Dec 05
PMID:Accuracy of DNA primase. 225 21

We had earlier characterized the nascent DNA synthesized in permeable cells of Bacillus subtilis in the presence of 5-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP as being substituted at its 5' end with a ribonucleotide moiety of the sequence pApG(pC)1-2 DNA. In this paper, we examine the origin and turnover of the DNA-linked ribonucleotide and its relationship to DNA replication. At least 50% of the RNA-linked nascent DNA chains served as guanylate acceptors when incubated with GTP and the eukaryotic capping enzyme, indicating the presence of 5'-terminal di- or triphosphate groups and suggesting that the RNA moiety is synthesized de novo and is not a degradation product. In nascent DNA produced without limitation of chain growth by dideoxyATP, the degree of terminal ribonucleotide substitution was reduced by 50%, consistent with a linkage between RNA primer removal and DNA chain growth. Such a relationship was demonstrated directly by examining the RNA primer content of nascent DNA synthesized in the absence of dideoxyATP as a function of DNA chain length. As the DNA size increased from 40 to 200 nucleotide residues, the extent of RNA substitution declined from 80% to nearly 0%. Endgroup analysis showed that the loss of RNA was accompanied by a gradual shift from predominantly adenylate residues to 5'-terminal guanylate, consistent with a stepwise removal of ribonucleotides from the 5' end. Evidence that the nascent mercurated DNA synthesized under our experimental conditions was indeed a replicative intermediate came from the study of the time course of DNA chain growth and pulse-chase experiments. In the presence of the DNA ligase inhibitor NMN, mercurated DNA accumulated in two size classes with average length of approximately 750 and 8000 nucleotide residues, presumably representing the mature size of intermediates in discontinuous DNA synthesis. Comparison with the DNA size range at which the loss of the 5'-terminal RNA moiety occurred (40 to 200 residues) indicated that the processing of RNA primers occurred at an early stage during DNA chain elongation, and that moderate size intermediates in discontinuous DNA replication (greater than 200 nucleotides) have already lost their RNA primers.
J Mol Biol 1985 Nov 20
PMID:Origin and degradation of the RNA primers at the 5' termini of nascent DNA chains in Bacillus subtilis. 241 6

Small RNAs were studied in the cell-free system containing cytosol and nucleoside triphosphates. The specific incorporation of NMPs into small RNAs was shown. While AMP and CMP were incorporated predominantly into tRNA, UMP was incorporated into 4.5S, 5S, and U1 RNAs. GMP was not incorporated into small RNAs in this system. The data suggest that maturation of 4.5S, 5S, and U1 RNAs involves post-transcriptional addition of uridylate residues at their 3'-ends.
Mol Biol (Mosk)
PMID:[Template-independent incorporation of nucleotides into low-molecular weight RNA in a cell-free system]. 243 Jan 71

1. Excitatory amino acids (EAA) can activate second messenger systems in addition to a direct gating of ion channels. A discrete coupling between novel EAA receptor subtypes and second messenger systems has been previously proposed. 2. EAAs have been suggested to activate both adenylate and guanylate cyclases and also to induce phosphoinositide (PI) turnover. The increased PI turnover was observed in both central neurons and glia, and a "quisqualate-type" receptor has been most frequently involved, which may differ from the quisqualate receptor previously defined by electrophysiological studies. 3. The roles of EAA-induced calcium influx into neurons and raised intracellular calcium levels are discussed regarding the activation of phosphoinositide turnover. 4. This review examines the data supporting a link between EAA receptors and second messengers and considers whether there is any need for adopting new EAA receptor subtypes. Also, the use of the Xenopus laevis oocyte for expressing EAA receptors and studying any putative links to second messenger systems is discussed.
Cell Mol Neurobiol 1989 Jun
PMID:Excitatory amino acids: the involvement of second messengers in the signal transduction process. 247 89

The enzyme ribonuclease T1 (RNase T1) isolated from Aspergillus oryzae was cocrystallized with the specific inhibitor guanylyl-2',5'-guanosine (2',5'-GpG) and the structure refined by the stereochemically restrained least-squares refinement method to a crystallographic R-factor of 14.9% for X-ray data above 3 sigma in the resolution range 6 to 1.8 A. The refined model consists of 781 protein atoms, 43 inhibitor atoms in a major site and 29 inhibitor atoms in a minor site, 107 water oxygen atoms, and a metal site assigned as Ca. At the end of the refinement, the orientation of His, Asn and Gln side-chains was reinterpreted on the basis of two-dimensional nuclear magnetic resonance data. The crystal packing and enzyme conformation of the RNase T1/2',5'-GpG complex and of the near-isomorphous RNase T1/2'-GMP complex are comparable. The root-mean-square deviation is 0.73 A between equivalent protein atoms. Differences in the unit cell dimensions are mainly due to the bound inhibitor. The 5'-terminal guanine of 2',5'-GpG binds to RNase T1 in much the same way as in the 2'-GMP complex. In contrast, the hydrogen bonds between the catalytic center and the phosphate group are different and the 3'-terminal guanine forms no hydrogen bonds with the enzyme. This poor binding is reflected in a 2-fold disorder of 2',5'-GpG (except the 5'-terminal guanine), which originates from differences in the pucker of the 5'-terminal ribose. The pucker is C2'-exo for the major site (2/3 occupancy) and C1'-endo for the minor site (1/3 occupancy). The orientation of the major site is stabilized through stacking interactions between the 3'-terminal guanine and His92, an amino acid necessary for catalysis. This might explain the high inhibition rate observed for 2',5'-GpG, which exceeds that of all other inhibitors of type 2',5'-GpN. On the basis of distance criteria, one solvent peak in the electron density was identified as metal ion, probably Ca2+. The ion is co-ordinated by the two Asp15 carboxylate oxygen atoms and by six water molecules. The co-ordination polyhedron displays approximate 4m2 symmetry.
J Mol Biol 1989 Apr 05
PMID:Three-dimensional structure of ribonuclease T1 complexed with guanylyl-2',5'-guanosine at 1.8 A resolution. 254 Dec 56

The crystals of a complex between ribonuclease Ms, the extracellular ribonuclease from Aspergillus saitoi, and 3'-guanylic acid were obtained from 2-methyl-2,4-pentanediol solution by vapor diffusion technique in the hanging drop mode. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with dimensions a = 47.0 A, b = 62.8 A, c = 37.9 A. The crystals diffract strongly up to at least 2.0 A resolution.
J Mol Biol 1989 Jun 20
PMID:Crystallization of a complex between ribonuclease Ms and 3'-guanylic acid. 254 76


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