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Query: EC:6.1.1.20 (phenylalanyl-tRNA synthetase)
 358 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kinetics of the affinity modification of phenylalanyl-tRNA synthetase from E. coli MRE-600 with chb-tRNA was used for investigation of copling between the binding sites of tRNA and other ligands. It was shown that ATP, phenylalanine and their mixture do not change the efficiency of complex formation but decrease specifically the rate of enzyme alkylation. L-Tyrosine and L-valine do not influence the enzyme alkylation. ATP is more effective protector than L-phenylalanine. In the presence of both ATP and phenylalanine the enzyme alkylation is excluded. The possibilities of this method for studying the coupling between binding sites are discussed.
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PMID:[Affinity modification of phenylalanyl-tRNA-synthetase in the presence of ligands]. 77 90

A procedure for the simultaneous isolation of seryl- and phenylalanyl-tRNA synthetase from yeast is described. In addition some other synthetases as well as tRNA nucleotidyltransferase can be obtained in an enriched state. The isolated seryl- and phenylalanyl-tRNA synthetases were compared to earlier preparations with respect to purity, specific activity, and structure. Previous investigations with fluorescence spectroscopy and kinetic methods were complemented and extended by experiments on the specificity of aminoacylation and on the isolation, by sucrose gradient centrifugation, of complexes between synthetase and tRNA or tRNA fragments. A protection of synthetases against inactivation by addition of substrates was observed. The dissociation of seryl-tRNA synthetase, at low concentrations, into monomer subunits was investigated by chemical modification with bifunctional reagents and by kinetic experiments. By modification of SH-groups fluorescent dyes were incorporated into both, seryl- and phenylalanyl-tRNA synthetase which retained most of their activity. The binding of tRNAPhe to phenylalanyl-tRNA synthetase which had been modified with pyrene maleimid was followed by fluorescence intensity measurements.
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PMID:[Isolation and characterization of seryl- and phenylalanyl-tRNA synthetase from yeast (author's transl)]. 78 43

Incorrect tRNA aminoacylation reactions are characterized by very slow reaction rates and by the fact that in most cases they are incomplete. In a previous study some of us explained the incompleteness of the correct aminoacylation reactions of tRNA, which can be encountered under certain experimental conditions (for instance low enzyme concentration or high ionic strength) by an equilibrium between the aminoacylation and the deacylation reactions [J. Bonnet and J.P. Ebel (1972) Eur. J.Biochem. 31, 335-344]. In the present report we bring evidence that the incorrect valylation of yeast tRNAfMet by yeast valyl-tRNA synthetase studied under standard experimental conditions, can also be described by a kinetic rate law including the rate equations of the aminoacylation and of the various deacylation reactions. In particular we show that the incomplete mischarging plateaus reflect the existence of an equilibrium between the valylation reaction on the one hand and the spontaneous and enzymic deacylation of valyl-tRNAfMet and the reverse of the valylation reaction on the other hand. However, when the valyl-tRNA synthetase concentration is not very high the reverse reaction of the amino-acylation does not play a predominant part in the establishment of the plateau. These interpretations have been extended to other mischarging systems: valylation of yeast tRNAPhE by yeast valyl-tRNA synthetase and mischarging of tRNAfMet and tRNA2Val from yeast by yeast phenylalanyl-tRNA synthetase. Unusual mischarging kinetics have been discussed. Furthermore, and as in correct systems, we found that during the mischarging of tRNAfMet one ATP is hydrolyzed per tRNA charged with valine. We conclude that the correct and the incorrect amino-acylation of tRNA behave kinetically in a similar way.
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PMID:Interpretation of tRNA-mischarging kinetics. 79 46

The studies described indicate that the UV bleached mutant, Euglena gracilis W3BUL does not serve as a suitable cytoplasmic control for the phenylalanyl-tRNA synthetase system. Chromatography of wild-type E. gracilis on Sephadex G100 revealed three peaks of activity identified as the chloroplastic, cytoplasmic and mitochondrial enzymes. The chloroplastic activity was greater in log than in stationary phase cells and was the only activity recovered from purified chloroplasts. Cell-free extracts of the achloroplstic mutant, E. gracilis W3BUL, contained wild-type levels of the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases. However, no chloroplastic synthetase was detected in the mutant extracts. Anomalies in the aminoacylation behavior of the W3BUL system were observed which suggest the possibility of a mutation affecting non-chloroplastic tRNAs in this UV-induced mutant. These anomalies significantly reduce the ability of the E. gracilis W3BUL mutant to serve as a cytoplasmic control in the phenylalanyl-tRNA synthetase system.
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PMID:A consideration of Euglena gracilis W3BUL as a cytoplasmic control for the wild-type phenylalanyl-tRNA synthetase system. 81 46

A response to: "A consideration of Euglena gracilis W3BUL as a cytoplasmic control for the wild-type phenylalanyl-tRNA synthetase system" and "A reinvestigation of the sites of transcription and translation of Euglena chloroplastic phenylalanyl-tRNA synthetase" by J. L. Lesiewicz and D. S. Herson.
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PMID:Comments on the translational and transcriptional origin of Euglena chloroplastic aminoacyl-tRNA synthetases. 82 72

Formation of short, double-stranded RNA helices by the complementary oligoribonucleotides GpApGpC, GpCpUpC, and ApGpCpUpC was established by the temperature-dependent changes in uv hypochromicity and circular dichroism spectra. These studies provide additional thermodynamic data on duplex formation. Duplexes (formula: see text) are especially significant as they: (1) correspond to a natural sequence, the dihydrouridine loop neck region, common to several tRNA molecules; (2) correspond to a proposed, partial recognition site for yeast phenylalanyl-tRNA ligase (EC 6.1.1.20); and (3) represent the first demonstration of a duplex with four base pairs, one of which is an A--U pair. These duplexes should serve as models for studies on tRNA-ligase interaction.
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PMID:Duplex formation of complementary oligoribonucleotides corresponding to the dihydrouridine loop neck region of several transfer ribonucleic acids. 85 86

Phenylalanyl-tRNA and seryl-tRNA synthetase protect strongly though not completely their cognate tRNAs against nuclease attack, as had been shown previously. In an investigation of the mechanism of protection it was demonstrated that the low susceptibility of phenylalanyl-tRNA-synthetase x tRNA-Phe complexes to nucleases is due to free tRNA present in equilibrium with synthetase. The equilibrium can be shifted by an excess of synthetase or by dilution of the complex. It therefore appears that synthetase competes with the nuclease for free tRNA. Degradation of the complex is low, however, because under the conditions of partial digestion the synthetase has a greater affinity for the tRNA than does the nuclease. Fragmented tRNAs, as they are formed during partial nuclease digestion, bind to synthetase to different degrees. tRNA-Phe with a lesion in the dihydrouridine loop binds very poorly whereas a nick in the anticodon loop reduces the strength of binding to a much lesser extent. In a systematic study of the stoichiometry of protection it was confirmed that under standard conditions one phenylalanyl-tRNA synthetase protects one tRNA-Phe and one seryl-tRNA synthetase two tRNA-Ser molecules against nuclease attack. Under certain conditions, however, (concentration of the complex higher than 10 mu-M, or alternately in buffers of low ionic strength) it is observed that phenylalanyl-tRNA synthetase binds up to 1.6 molecules tRNA-Phe. In the serine system, these special conditions do not affect the binding properties of seryl-tRNA synthetase.
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PMID:Nuclease digestion of synthetase x tRNA complexes. 109 70

Complexes between tRNAPhe (yeast), tRNASer (yeast) and tRNATyr (Escherichia coli) and their cognate aminoacyl-tRNA synthetases have been studied by sedimentation velocity runs in an analytical ultracentrifuge. The amount of complex formation was determined by the absorption and the sedimentation coefficients of the fast-moving boundary in the presence of excess tRNA or excess synthetase respectively. The same method has been applied to unspecific combinations of tRNAs and synthetases. Inactive material of tRNA or synthetase does not influence the results. 1. Two moles of tRNAPhe can be bound to one mole of phenylalanyl-tRNA synthetase with a binding constant greater than 10(6) M-1. The binding constants for both tRNAs are very similar; the binding sites are independent of each other. Omission of Mg2+ does not prevent binding. 2. Two moles of tRNASer can be bound to one mole of Seryl-tRNA synthetase; the binding of the first and second tRNA is non-equivalent, K1 greater than 10(6) M-1, K2 is determined to be 1.3 X 10(5) M-1 at pH 7.2. Omission of Mg2+ prevents complex formation. 3. Tyrosyl-tRNA synthetase behaves very similarly to seryl-tRNA synthetase. The binding constant for the weakly bound tRNA is 2.3 X 10(5) M-1 at pH 7.2, and 2.5 X 10(6) M-1 at pH 6.0. No complexes are observed in the absence of Mg2+. 4. Unspecific binding was only obtained with phenylalanyl-tRNA synthetase. It binds tRNASer (yeast), tRNAAla (yeast) and tRNATyr (E. coli) with a binding constant about 100 times lower compared to its cognate tRNA. The binding data are discussed with respect to the tertiary structure of the tRNAs, the subunit structure of the synthetases and the possible physical basis for the non-equivalence of binding sites.
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PMID:Equivalent and non-equivalent binding sites for tRNA on aminoacyl-tRNA synthetases. 110 Mar 84

The inhibitory effect of aurintricarboxylic acid (ATA) on phenylalanyl-tRNA synthetase is demonstrated in the rabbit-reticulocyte system. This inhibition is not specific to ATA; other triphenylmethane derivatives are also potent inhibitors of this enzyme reaction. The site of inhibitory action is in the enzyme itself, not in the tRNA molecule. It thus appears that the skeletal structure of ATA, not the side chains, is responsible for its inhibitory action, and that ATA is a nonspecific inhibitor of the reactions involving polynucleotides.
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PMID:Effect of triphenylmethane derivatives on cell-free macromolecular synthesis. I. Aminoacyl-tRNA synthetase. 112 8

Thyroxine and analogues inhibit rat liver aminoacyl-tRNA synthetase activity for phenylalanine and tyrosine. A high yield purification of the major cytoplasmic form of phenylalanyl-tRNA synthetase (C1) and its characterization is reported. Polyribosome-bound and other sedimentable forms are found to be indistinguishable from soluble enzyme by immunoprecipitation. Mitochondrial phenylalanyl-tRNA synthetase (M) and cytoplasmic activity (C2) resistant to anti-C1 antibody have been partially purified and characterized. Tissue levels of the three forms are estimated at 22, 1.8, and 4.1 units/g of liver for C1, C2, and M, respectively [1 unit = 1 nmol of Phe-tRNA/min, 30 degrees C]. Charging capability toward rat liver and yeast tRNA, kinetic parameters, and physical properties are compared. Only enzyme C1 is hormone inhibited [K1 = 4 x 10(-6) M for triiodothyronine]. The data indicata that C2 and M are not structurally related to C1; C2 may represent an independent cytoplasmic pool of M. Implications of C1 inhibition in relation to effects on liver protein synthesis are discussed.
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PMID:Phenylalanyl-tRNA synthetases of rat liver: differential effects of thyroid hormone. 124 20


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