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

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Query: EC:3.6.1.25 (triphosphatase)
1,529 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The partially purified preparation of messenger RNA guanylyltransferase from Artemia salina contains, as in the case of the rat liver enzyme (Yagi, Y., Mizumoto, K., and Kaziro, Y. (1983) EMBO J. 2, 611-615), the RNA 5'-triphosphatase activity which specifically removes the gamma-phosphoryl group from the 5'-triphosphoryl end of the newly synthesized mRNA molecule. The enzyme consists of a single polypeptide chain of Mr = 73,000 and forma a covalent enzyme-GMP complex as an intermediate for the guanylyltransferase reaction. Upon limited hydrolysis with trypsin, the enzyme-[32P]GMP complex is converted to a smaller 32P-containing fragment of Mr = 44,000. When the free enzyme, not complexed with GMP, is digested with trypsin under the same condition as above, the digests retain almost full activities of both guanylyltransferase and RNA 5'-triphosphatase and can form an enzyme-[32P]GMP complex of the size of Mr = 44,000 on incubation with [alpha-32P]GTP. Functional domains harboring the activities of guanylyltransferase and RNA 5'-triphosphatase are separated by gel filtration on a Sephacryl S-200 column at positions corresponding to Mr = 44,000 and 20,000, respectively. They can be separated completely from each other by CM-Sephadex column chromatography. While the native, undigested enzyme can transfer the GMP moiety to mRNA molecules with either triphosphoryl (pppN-) or diphosphoryl (ppN-)5'terminal, the purified Mr = 44,000 domain with the guanylyltransferase activity can utilize only the latter as an acceptor.
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PMID:Limited tryptic digestion of messenger RNA capping enzyme from Artemia salina. Isolation of domains for guanylyltransferase and RNA 5'-triphosphatase. 614 51

The in vitro system of RNA transport containing isolated nuclei of Djungarian hamster cells transformed by SV-40 virus was studied. A functional test for cytoplasmic contaminations of the nuclei was proposed. The release of the newly synthesized RNA was found to be dependent on the duration of incubation, temperature and pH of the incubation medium as well as on the presence of spermine, spermidine, dithiothreitol, Mg2+, EDTA, exogenous RNA, nucleoside triphosphates and cytosol. The results obtained indicate that RNA release is an active process with activation energy of 12 kcal/mol. ATP, GTP, CTP and UTP have equal ability to serve as energy sources for the release of RNA. The nucleoside triphosphatase activity of the nuclei was the same in the presence of these four nucleoside triphosphates.
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PMID:[RNA transport from isolated nuclei of cultured Djungarian hamster cells]. 617 80

Adenosine triphosphatase activity which is Mg2+-dependent and stimulated by submicromolar concentrations of Ca2+ (as Ca . ATP) was identified in the total particulate fraction of rat pancreatic acini. Half-maximal activity (V0.5) is obtained at 100.1 +/- 6 nM Ca . ATP with a Hill coefficient of 2.2 +/- 0.1 (mean +/- S.E.; n = 4). Maximal activity was 75 +/- 19 pmol of Pi released from ATP minute-1 microgram of membrane protein-1 (mean +/- S.E.; n = 7). High affinity Ca2+-ATPase activity was unaffected by ouabain, Na+, K+, La3+, and added calmodulin. Activity was slightly reduced by ruthenium red (0.1 mM) and by oligomycin (80 micrograms/ml) but was reduced almost 50% by the phenothiazine derivative fluphenazine in a dose-related and Ca2+-dependent manner. Hydrolysis of p-nitrophenyl phosphate was 9% of the rate of ATP hydrolysis and was independent of Ca2+ concentration. However, ADP, GTP, UTP, and ITP were hydrolyzed at 76-93% the rate that ATP was hydrolyzed with V0.5 values and Hill coefficients similar to those of Ca . ATP. We conclude that rat pancreatic acini contain an enzyme for active Ca2+ translocation: ATPase activity that is Mg2+-dependent and stimulated by submicromolar concentrations of Ca . ATP. Substrate hydrolysis appears to involve positive cooperative interactions of multiple ligand-binding sites and may be regulated in part by calmodulin.
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PMID:High affinity, calcium-stimulated adenosine triphosphatase activity in the particulate fraction of rat pancreatic acini. 623 78

A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzyme requires MgCl2 for activity, has an alkaline pH optimum, and specifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP. The GTP-PPi exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase) through successive chromatography steps on DEAE-cellulose, DNA-cellulose, and phosphocellulose. GTP-PPi exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PPi exchange, capping, and methylating activities co-sediment with an RNA triphosphatase activity and a nucleoside triphosphate phosphohydrolase activity as a 6.5 S multifunctional enzyme complex which contains two major polypeptides of 96,000 and 26,000 molecular weight. The characteristics of the various enzymatic reactions catalyzed by this complex are described. The GTP-PPi exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PPi exchange reaction to the mechanism of transguanylylation is considered.
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PMID:Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme). 625 74

Guanylyltransferase that catalyzes mRNA capping by the reaction, ppNpN + GTP----GpppNpN was purified from S. cerevisiae. The enzyme forms a nucleotidyl intermediate by phosphoamide linkage of GMP. Two guanylylated polypeptides of MR approximately 52,000 and 46,000 were obtained, the latter apparently by proteolysis of the larger component. Both forms transferred the covalently bound GMP to ppApG, yielding GpppApG. Dinucleoside tri- and tetraphosphates of the type Gp3N and Gp4N were also produced by using ribonucleoside 5'-di and triphosphates as acceptors. The purified yeast guanylyltransferase contained little or no RNA 5'-triphosphatase or methyltransferase.
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PMID:Synthesis of Gp4N and Gp3N compounds by guanylyltransferase purified from yeast. 632 12

(2'-5')-oligoadenylates bearing a 5'-terminal triphosphate or a 5'GTP-group inhibit the activity of a dinucleoside triphosphatase in rat liver nuclei thereby protecting mRNA against 5'-exonucleolytic degradation. (2'-5')-oligoadenylates, on the other hand, were known to enhance the activity of an endoribonuclease, RNaseF. Thus a synergistic effect may be assumed in vivo, i.e. cellular metabolism seems to be protected twice in virus-infected cells.
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PMID:Effect of 5'-terminated (2'-5')-oligoadenylates on cap degrading activities in rat liver nuclei. 663 82

GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of a GMP residue from GTP to the 5' end of RNA to form a cap structure identified as G(5')pppN-, has been isolated from HeLa cell nuclei. The enzyme has been purified approximately 1000-fold and separated by column chromatography (using DEAE-cellulose, phosphocellulose, Cibacron blue-agarose, and GTP-agarose) from a variety of other activities, including RNA triphosphatase and mRNA (guanine-7)methyltransferase. The reaction product was identified by its resistance to Penicillium nuclease and alkaline phosphatase, sensitivity to venom phosphodiesterase, and electrophoretic and chromatographic mobilities relative to authentic standards. Optimal enzyme activity was obtained at pH 7.5 in the presence of Mn2+ or Mg2+, GTP, and an appropriate acceptor polyribonucleotide. The enzyme was inhibited by elevated concentrations of salt and by sulfhydryl-binding reagents but was unaffected by S-adenosylmethionine or S-adenosylhomocysteine. A molecular weight of 48,500 was estimated by sucrose gradient centrifugation of purified enzyme.
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PMID:Purification and characterization of mRNA guanylyltransferase from HeLa cell nuclei. 735 12

Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, RNA (guanine-7) methyltransferase, and transcription termination factor activities. The protein is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The capping reaction entails transfer of GMP from GTP to the 5'-diphosphate end of mRNA via a covalent enzyme-(lysyl-GMP) intermediate. The active site is situated at Lys-260 of the D1 subunit within a sequence element, KxDG (motif I), that is conserved in the capping enzymes from yeasts and other DNA viruses and at the active sites of covalent adenylylation of RNA and DNA ligases. Four additional sequence motifs (II to V) are conserved in the same order and with similar spacing among the capping enzymes and several ATP-dependent ligases. The relevance of these common sequence elements to the RNA capping reaction was addressed by mutational analysis of the vaccinia virus D1 protein. Nine alanine substitution mutations were targeted to motifs II to V. Histidine-tagged versions of the mutated D1 polypeptide were coexpressed in bacteria with the D12 subunit, and the His-tagged heterodimers were purified by Ni affinity and phosphocellulose chromatography steps. Whereas each of the mutated enzymes retained triphosphatase, methyltransferase, and termination factor activities, six of nine mutant enzymes were defective in some aspect of transguanylylation. Individual mutations in motifs III, IV, and V had distinctive effects on the affinity of enzyme for GTP, the rate of covalent catalysis (EpG formation), or the transfer of GMP from enzyme to RNA. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a conserved structural basis for covalent nucleotidyl transfer.
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PMID:Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA. 756 75

Sedimentation and high performance liquid chromatography studies show that the functional DNA replication helicase of bacteriophage T4 (gp41) exists primarily as a dimer at physiological protein concentrations, assembling from gp41 monomers with an association constant of approximately 10(6) M-1. Cryoelectron microscopy, analytical ultracentrifugation, and protein-protein cross-linking studies demonstrate that the binding of ATP or GTP drives the assembly of these dimers into monodisperse hexameric complexes, which redissociate following depletion of the purine nucleotide triphosphatase (PuTP) substrates by the DNA-stimulated PuTPase activity of the helicase. The hexameric state of gp41 can be stabilized for detailed study by the addition of the nonhydrolyzable PuTP analogs ATP gamma S and GTP gamma S and is not significantly affected by the presence of ADP, GDP, or single-stranded or forked DNA template constructs, although some structural details of the hexameric complex may be altered by DNA binding. Our results also indicate that the active gp41 helicase exists as a hexagonal trimer of asymmetric dimers, and that the hexamer is probably characterized by D3 symmetry. The assembly pathway of the gp41 helicase has been analyzed, and its structure and properties compared with those of other helicases involved in a variety of cellular processes. Functional implications of such structural organization are also considered.
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PMID:The phage T4-coded DNA replication helicase (gp41) forms a hexamer upon activation by nucleoside triphosphate. 770 92

Guanosine triphosphatase activating protein (GAP) is an important modulator of p21ras (Ras)-dependent signal transduction in mammalian cells and in insulin-induced maturation of Xenopus oocytes. A synthetic octapeptide from the catalytic domain of GAP, residues 899-906 (F899VFLRLIC906), inhibited GAP-stimulated hydrolysis of GTP to GDP by Ras in an in vitro biochemical assay (IC50 = 12 microM). The peptide was assayed for its ability to block insulin- (Ras-dependent) and progesterone- (Ras-independent) induced maturation of stage VI Xenopus laevis oocytes, marked by germinal vesicle breakdown (GVBD). Microinjection of 50 pmol of the peptide inhibited insulin- but not progesterone-induced GVBD by 50%. A 7-residue peptide lacking F899, GAP(900-906)-NH2, failed to inhibit GAP-stimulated GTPase activity and did not block GVBD. Replacement of the cysteine residue at position 906 with methionine resulted in a peptide with prolonged inhibitory activity in the oocyte. Moreover, sequential replacement of specific L-amino acid residues with the corresponding D-amino acids produced a peptide with a two-fold increased half-life after injection into oocytes. None of the peptides tested affected progesterone induced GVBD, suggesting that the modifications did not result in loss of specificity. These studies show that (a) peptides that were able to inhibit GAP-stimulated Ras GTPase activity in vitro were also able to block Ras-dependent GVBD in oocytes, and (b) specific substitutions in these peptides can result in improved stability in oocytes.
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PMID:Ras-dependent maturation of Xenopus oocytes is blocked by modified peptides of GTPase activating protein (GAP). 778 68


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