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 effect of various cytokines [interleukin-3(IL-3), IL-6, IL-8, tumour necrosis factor-beta (TNF-beta)] on human neutrophils (PMN) was analysed with regard to the generation of leukotrienes and the involvement of guanosine triphosphate (GTP)-binding proteins (G proteins). Incubation of cytochalasin B-pretreated PMN with cytokines alone did not lead to a generation of leukotrienes. However, the cytokines affected the formyl-methionyl-leucyl-phenylalanine-(FMLP)-induced formation of leukotrienes in a time-dependent manner. Preincubation of the cells with the different cytokines for short periods (15 seconds at 37 degrees) enhanced the subsequent FMLP-induced leukotriene generation, whereas preincubation for prolonged times resulted in a reduced formation of leukotrienes. These results correlated with the respective G protein-associated guanosine triphosphatase (GTPase) activities within isolated membrane fractions. The present study indicates a modulation of the FMLP-induced leukotriene formation by diverse cytokines via interaction with the GTP-binding proteins.
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PMID:Cytokine-induced (interleukins-3, -6 and -8 and tumour necrosis factor-beta) activation and deactivation of human neutrophils. 131 95

The effects of neomycin on human neutrophils (PMN) were studied with respect to the generation of leukotrienes, the involvement of guanine triphosphate binding proteins (G proteins) and the polymerization of actin. Incubation of neutrophils with neomycin induced the generation of low amounts of leukotrienes. Co-incubation of neutrophils with neomycin and the direct G-protein activator sodium fluoride (NaF) resulted in an enhanced leukotriene formation at 0.5 mM neomycin and an inhibition at a concentration of 10 mM. Simultaneous incubation with neomycin and formyl-methionyl-leucyl-phenylalanine (FLMP) did not affect the FMLP-induced leukotriene formation. However, pretreatment of neutrophils with 10 mM neomycin followed by the addition of NaF or FMLP resulted in an enhanced generation of leukotrienes. Crude membrane fractions of PMN incubated with neomycin at different concentrations showed an enhanced (0.5 mM) as well as a reduced (10 mM) guanine triphosphatase activity. Furthermore, incubation of neutrophils with neomycin above a concentration of 0.5 mM led to the depolymerization of actin. The presented results show inhibitory and stimulatory effects of neomycin on various cell functions, which may reflect differences in transmembrane signalling.
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PMID:Neomycin induces stimulatory and inhibitory effects on leukotriene generation, guanine triphosphatase activity, and actin polymerization within human neutrophils. 153 91

In hippocampal slices, somatostatin 14 and its stable analog L363 [cyclo(Phe-Pro-Phe-D-Trp-Lys-Thr)] fail to modify muscarinic signal transduction mediated by stimulation of phosphoinositide breakdown, whereas somatostatin 14 mimics oxotremorine in inhibiting adenylate cyclase activity of hippocampal membranes. The simultaneous addition of somatostatin 14 and oxotremorine elicits a nonadditive convergent inhibition of adenylate cyclase activity. Both L363 and oxotremorine nonadditively stimulate a high-affinity guanosine 5'-triphosphatase activity of hippocampal membranes. This stimulation could be operative in mediating the convergent inhibition of adenylate cyclase activity elicited by the binding of specific ligands to somatostatin and muscarinic recognition sites present in hippocampal membranes. Because L363 competitively displaces muscarinic agonists fand antagonists from their specific recognition sites, one might infer that the two recognition sites interact functionally; that is, somatostatin reduces the efficacy of oxotremorine and/or vice versa.
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PMID:In rat hippocampus, somatostatin 14 and muscarinic receptor ligands modulate an adenylate cyclase belonging to a common domain of the receptor. 288 75

The mechanism of action of diphtheria toxin in an Escherichia coli cell-free protein-synthesizing system was examined. When the washed ribosomes were incubated with toxin before addition of messenger ribonucleic acid (RNA), peptide syntheses of (14)C-phenylalanine directed by polyuridylic acid or phage R17 RNA were strongly inhibited by a small amount of toxin. Whereas, if the soluble fraction (105,000 x g supernatant fraction) was preincubated with toxin, no effect of toxin occurred either on the induced protein synthesis or on the activity of guanosine triphosphatase even in the presence of nicotinamide adenine dinucleotide. The binding of (3)H-formylmethionyl-transfer RNA to E. coli ribosomes directed by either R17 RNA or trinucleotide AUG was also decreased by toxin. These findings suggest that diphtheria toxin may prevent the binding of messenger RNA by successfully competing with the AUG for ribosomal binding sites. Sucrose-density gradient studies support this concept by showing the decrease in binding of (3)H-labeled R17 RNA to E. coli ribosomes exposed to toxin.
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PMID:Inhibitory effect of diphtheria toxin on amino acid incorporation in Escherichia coli cell-free system. 431 20

1. Polyribosomes and ribosomal subunits from rat liver were adsorbed on a cellulosic ion-exchange adsorbent, freeze-dried and extracted with organic solvents. The activity of extracted particles in peptide elongation was tested in the presence of purified peptideelongation factors. 2. Chloroform-methanol mixture (2:1, v/v) extracted 1.87+/-0.15 pmol of cholesteryl 14-methylhexadecanoate/pmol of the smaller ribosomal subunit and 0.92+/-0.11 pmol/pmol of the larger subunit. 3. In the presence of transferase I, extracted polyribosomes and 40S subunits bound more phenylalanyl-tRNA than did control non-extracted particles. The same binding as in control mixtures was obtained with extracted particles supplemented with cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. The polymerization of phenylalanine was greatly decreased with extracted polyribosomes and subunits and addition of the cholesteryl ester could not fully restore the original activity. 5. Extraction significantly decreased the activity of the P site of peptidyl transferase and normal activity was recovered after the addition of the ester. The A site of peptidyl transferase in extracted polyribosomes showed an increased activity when compared with non-extracted polyribosomes. 6. Cholesteryl 14-methylhexadecanoate apparently affects the function of the ribosomal A site and peptidyl transferase site and probably also that of the guanosine triphosphatase site and P site. The presence of different amounts of the ester in polyribosomes may be one of the mechanisms modulating peptide elongation at the ribosomal level.
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PMID:Influence of cholesteryl 14-methylhexadecanoate on some ribosomal functions required for peptide elongation. 459 29

The Escherichia coli strain D2216 contains a kirromycin-resistant elongation factor Tu [EF-Tu(D2216); Fischer, E., Wolf, H., Hantke K., & Parmeggiani, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4341-4345]. This stain grows much more slowly than wild-type E. coli strains and contains less than half the amount of EF-Tu. On isoelectric focusing, the whole cell lysate of strain D2216 as well as pure, crystalline EF-Tu(D2216) comprises only a single species indistinguishable from wild-type EF-Tu. In poly(uridylic acid)- [poly(U)] directed poly(phenylalanine) synthesis, enzymatic binding of aminoacyl transfer ribonucleic acid to the ribosome, and susceptibility to trypsin digestion, EF-Tu(D2216) behaves similarly to the EF-Tu from wild-type strains. Kirromycin, which increases the sensitivity to trypsinization of wild-type EF-Tu, has no effect on mutant EF-Tu. In poly(U)-directed poly(phenylalanine) synthesis, partially trypsinized EF-Tu(D2216) displays a 7-fold reduction of its kirromycin resistance as compared to the intact EF-Tu(D2216). This is approximately 300 times less sensitive to the antibiotic than wild-type EF-Tu. The EF-Tu(D2216), purified and crystallized, exhibits a guanosine 5'-triphosphatase activity in the absence of any other physiological effector or kirromycin. This activity is not a contaminant, since it can be selectively stimulated by ribosomes and is inactivated by temperature exactly in the same way as the guanosine 5'-diphosphate binding activity of Ef-Tu(D2216). We conclude that, as consequence of the mutation, the catalytic center of EF-Tu(D2216)-dependent guanosine 5'-triphosphate hydrolysis undergoes spontaneous activation.
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PMID:Characterization of a kirromycin-resistant elongation factor Tu from Escherichia coli. 701 93

The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that is required for transcription of viral late genes. This polymerase is composed of four equimolar subunits, LEF-8, LEF-4, LEF-9, and p47. The LEF-4 subunit has guanylyltransferase activity, suggesting that baculoviruses may encode a full complement of capping enzymes. Here we show that LEF-4 is a bifunctional enzyme that hydrolyzes the gamma phosphates of triphosphate-terminated RNA and also hydrolyzes ATP and GTP to the respective diphosphate forms. Alanine substitution of five residues previously shown to be essential for vaccinia virus RNA triphosphatase activity inactivated the triphosphatase component of LEF-4 but not the guanylyltransferase domain. Conversely, mutation of the invariant lysine in the guanylyltransferase domain abolished the guanylyltransferase activity without affecting triphosphatase function. We also investigated the effects of substituting phenylalanine for leucine at position 105, a mutation that results in a virus that is temperature sensitive for late gene expression. We found that this mutation had no significant effect on the ATPase or guanylyltransferase activity of LEF-4 but resulted in a modest decrease in RNA triphosphatase activity.
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PMID:The LEF-4 subunit of baculovirus RNA polymerase has RNA 5'-triphosphatase and ATPase activities. 981 39

Human and fission yeast cDNAs encoding mRNA (guanine-N7) methyltransferase were identified based on similarity of the human (Hcm1p; 476 amino acids) and Schizosaccharomyces pombe (Pcm1p; 389 amino acids) polypeptides to the cap methyltransferase of Saccharomyces cerevisiae (Abd1p). Expression of PCM1 or HCM1 in S. cerevisiae complemented the lethal phenotype resulting from deletion of the ABD1 gene, as did expression of the NH2-terminal deletion mutants PCM1(94-389) and HCM1(121-476). The CCM1 gene encoding Candida albicans cap methyltransferase (Ccm1p; 474 amino acids) was isolated from a C. albicans genomic library by selection for complementation of the conditional growth phenotype of S. cerevisiae abd1-ts mutants. Human cap methyltransferase was expressed in bacteria, purified, and characterized. Recombinant Hcm1p catalyzed quantitative S-adenosylmethionine-dependent conversion of GpppA-capped poly(A) to m7GpppA-capped poly(A). We identified by alanine-scanning mutagenesis eight amino acids (Asp-203, Gly-207, Asp-211, Asp-227, Arg-239, Tyr-289, Phe-291, and Phe-354) that are essential for human cap methyltransferase function in vivo. All eight residues are conserved in other cellular cap methyltransferases. Five of the mutant human proteins (D203A, R239A, Y289A, F291A, and F354A) were expressed in bacteria and found to be defective in cap methylation in vitro. Concordance of mutational effects on Hcm1p, Abd1p, and vaccinia capping enzyme underscores a conserved structural basis for cap methylation in DNA viruses, yeast, and metazoans. This is in contrast to the structural and mechanistic divergence of the RNA triphosphatase components of the yeast and metazoan capping systems. Nevertheless, we demonstrate that the entire three-component yeast capping apparatus, consisting of RNA 5'-triphosphatase (Cet1p), RNA guanylyltransferase (Ceg1p), and Abd1p could be replaced in vivo by the two-component mammalian apparatus consisting of a bifunctional triphosphatase-guanylyltransferase Mce1p and the methyltransferase Hcm1(121-476)p. Isogenic yeast strains with fungal versus mammalian capping systems should facilitate rational screens for antifungal drugs that target cap formation in vivo.
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PMID:Characterization of human, Schizosaccharomyces pombe, and Candida albicans mRNA cap methyltransferases and complete replacement of the yeast capping apparatus by mammalian enzymes. 1034 20

Saccharomyces cerevisiae Cet1p is the prototype of a family of metal-dependent RNA 5'-triphosphatases/NTPases encoded by fungi and DNA viruses; the family is defined by conserved sequence motifs A, B, and C. We tested the effects of 12 alanine substitutions and 16 conservative modifications at 18 positions of the motifs. Eight residues were identified as important for triphosphatase activity. These were Glu-305, Glu-307, and Phe-310 in motif A (IELEMKF); Arg-454 and Lys-456 in motif B (RTK); Glu-492, Glu-494, and Glu-496 in motif C (EVELE). Four acidic residues, Glu-305, Glu-307, Glu-494, and Glu-496, may comprise the metal-binding site(s), insofar as their replacement by glutamine inactivated Cet1p. E492Q retained triphosphatase activity. Basic residues Arg-454 and Lys-456 in motif B are implicated in binding to the 5'-triphosphate. Changing Arg-454 to alanine or glutamine resulted in a 30-fold increase in the K(m) for ATP, whereas substitution with lysine increased K(m) 6-fold. Changing Lys-456 to alanine or glutamine increased K(m) an order of magnitude; ATP binding was restored when arginine was introduced. Alanine in lieu of Phe-310 inactivated Cet1p, whereas Tyr or Leu restored function. Alanine mutations at aliphatic residues Leu-306, Val-493, and Leu-495 resulted in thermal instability in vivo and in vitro. A second S. cerevisiae RNA triphosphatase/NTPase (named Cth1p) containing motifs A, B, and C was identified and characterized. Cth1p activity was abolished by E87A and E89A mutations in motif A. Cth1p is nonessential for yeast growth and, by itself, cannot fulfill the essential role played by Cet1p in vivo. Yet, fusion of Cth1p in cis to the guanylyltransferase domain of mammalian capping enzyme allowed Cth1p to complement growth of cet1Delta yeast cells. This finding illustrates that mammalian guanylyltransferase can be used as a vehicle to deliver enzymes to nascent pre-mRNAs in vivo, most likely through its binding to the phosphorylated CTD of RNA polymerase II.
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PMID:Mutational analyses of yeast RNA triphosphatases highlight a common mechanism of metal-dependent NTP hydrolysis and a means of targeting enzymes to pre-mRNAs in vivo by fusion to the guanylyltransferase component of the capping apparatus. 1050 29

The N-formyl peptide receptor is a G protein-coupled transmembrane receptor involved in stimulating a variety of differential responses in neutrophils including chemotaxis, degranulation, superoxide production, transcriptional activation, and actin reorganization. Although it is known that N-formyl-Met-Leu-Phe induces actin reorganization, the sequence of events from the receptor to the actin cytoskeleton is not well characterized. To study the signaling pathway from the N-formyl peptide receptor to the actin cytoskeleton, we developed a model system utilizing microinjection techniques with a nonhematopoietic cell line. An expression vector coding for the N-formyl peptide receptor was microinjected into porcine aortic endothelial cells and stimulated with N-formyl-Met-Leu-Phe to induce actin reorganization and membrane ruffling. The receptor-mediated signal was blocked by pertussis toxin and by a dominant negative Rac-N17, indicating the involvement of G(i)alpha subunit and the small guanosine triphosphatase Rac, respectively. Moreover, Gbetagamma subunits and membrane targeted forms of phosphatidylinositol (PI) 3-kinase alpha were sufficient to induce similar actin reorganization, and coexpression of various mutants of PI 3-kinase with the N-formyl peptide receptor identified a link to class Ia PI-3 kinase-mediated actin reorganization.
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PMID:N-Formyl peptide receptor ligation induces rac-dependent actin reorganization through Gbeta gamma subunits and class Ia phosphoinositide 3-kinases. 1084 92


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