scholarly journals Effects of liver regeneration on tRNA contents and aminoacyl-tRNA synthetase activities and sedimentation patterns

1986 ◽  
Vol 236 (1) ◽  
pp. 163-169 ◽  
Author(s):  
U Del Monte ◽  
S Capaccioli ◽  
G Neri Cini ◽  
R Perego ◽  
R Caldini ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Cultured Cells ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Regenerating Liver ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Acceptor Capacity ◽  
Increased Activity

The tRNA content and aminoacyl-tRNA synthetases of regenerating liver in the phase of rapid growth were compared with those of livers from both intact and sham-operated rats. At 48 h after hepatectomy, the amount of active tRNA (called ‘total acceptor capacity’) is significantly higher in regenerating liver than in control livers, owing to a general, possibly not uniform, increase in the various tRNA families, which suggests that it may contribute to the increased protein synthesis and to decreased protein degradation as well. The activities of most, but not of all, aminoacyl-tRNA synthetases in cell sap of regenerating liver tend to be greater than normal. Increased activity of histidyl-tRNA synthetase fits in with the possibility that the mechanisms that control the rate of protein degradation through aminoacylation of tRNAHis in cultured cells [Scornik (1983) J. Biol. Chem. 258, 882-886] also operate in the liver and play a role in regeneration. Sedimentation analysis of cell sap in sucrose density gradients shows a shift of prolyl-tRNA synthetase activity toward the high-Mr form in regenerating liver. This change might be related to the positive protein balance and to growth in vivo, since it is also observed in the anaplastic Yoshida ascites hepatoma AH 130.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Polyribosomal and particulate distribution of lysyl- and phenylalanyl-transfer ribonucleic acid synthetases

1974 ◽  
Vol 143 (1) ◽  
pp. 191-195 ◽  
Author(s):  
Gale Moline ◽  
Arnold Hampel ◽  
M. Duane Enger
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Trna Synthetase ◽  
Differential Sensitivity ◽  
Synthetase Activity ◽  
Ribosomal Subunits ◽  
Ovary Cells ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Particulate Distribution

1. Only two aminoacyl-tRNA synthetases from Chinese hamster ovary cells are found associated with ribosomes and polyribosomes. 2. Phenylalanyl-tRNA synthetase activity is found with the 60S subunit, 80S monoribosome and individual polyribosomes. An additional 15S form of the enzyme is also seen. 3. Lysyl-tRNA synthetase activity is found in a form of about 20S and associated with ribosomal subunits and polyribosomes. The ribosomal subunits having lysyl-tRNA synthetase activity are about 6S larger than the bulk of the ribosomal subunits. 4. The lysyl- and phenylalanyl-tRNA synthetases found in different complexes have differential sensitivity to EDTA and centrifugation properties.

scholarly journals Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

Genes ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 473 ◽  
Author(s):  
Takuya Umehara ◽  
Saori Kosono ◽  
Dieter Söll ◽  
Koji Tamura
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Lysine Acetylation ◽  
Trna Synthetases ◽  
E Coli ◽  
Domains Of Life ◽  
The Impact

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.

A Genetic Study of Erythrocyte Arginine-tRNA Synthetase Activity in Man

1977 ◽  
Vol 26 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Sylvia A. McCune ◽  
P. L. Yu ◽  
Walter E. Nance
Keyword(s):  
Enzyme Activity ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Trna Synthetases ◽  
Automated Assay ◽  
Aminoacyl Trna Synthetases ◽  
Assay Procedure ◽  
Normal Human ◽  
Genetically Determined ◽  
Mz Twins

To search for evidence of genetic variation among the aminoacyl-tRNA synthetases, a semi-automated assay procedure employing a Technicon Auto Analyzer was used to measure erythrocyte arginine-tRNA synthetase activity in samples obtained from normal human twins of various ages. Variation in enzyme activity within the older DZ twins was five times that of the MZ twins suggesting the existence of genetically determined variation in enzyme activity. Higher enzyme activity was observed in newborn DZ unlike-sexed twins than in like-sexed twins of either zygosity. Possible explanations for this observation are discussed.

scholarly journals A novel pathway for the conversion of homocysteine to methionine in eukaryotes

1997 ◽  
Vol 328 (1) ◽  
pp. 165-170 ◽  
Author(s):  
M. Celia ANTONIO ◽  
C. Marta NUNES ◽  
Helga REFSUM ◽  
K. Abraham ABRAHAM
Keyword(s):  
Amino Acid ◽  
Crude Extract ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Enzyme Complex ◽  
Homocysteine Thiolactone ◽  
Initiator Trna ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Reticulocyte Lysates

Activation of amino acid homocysteine was compared with that of methionine in rabbit crude liver extracts and purified multi-enzyme complex of aminoacyl-tRNA synthetases. Activation was studied by measuring the incorporation of radioactive amino acid into unlabelled trichloroacetic-acid insoluble materials in the absence of protein synthesis. Homocysteine synthetase activity was found in the crude extract and in the purified multi-enzyme complex of aminoacyl-tRNA synthetases. On a molar basis, the activation of methionine by the crude extract was five times higher than the activation of homocysteine. There was a partial loss of Hcy-tRNA synthetase activity in the purified multi-enzyme complex. Preliminary reconstitution experiments indicated a requirement for an additional factor for Hcy-tRNA synthetase activity. TLC of the amino acid released from tRNA charged with [14C]homocysteine, revealed radioactivity in homocysteine, methionine and homocysteine thiolactone, indicating a conversion of tRNA-attached homocysteine to methionine. Total tRNA was separated on a benzoylated cellulose column into a fraction enriched in initiator tRNA and a methionine-accepting, but initiator tRNA-deficient, fraction. Homocysteine-accepting activity was present only in the initiator tRNA-enriched fraction. Based on the above data we propose that homocysteine activation in reticulocyte lysates, reported previously, also occurs in liver. Activated homocysteine is attached to initiator tRNA and then converted to methionine by a methylating enzyme. In the absence of methylation, tRNA-attached homocysteine is hydrolysed to produce homocysteine thiolactone.

scholarly journals Bacterial Aspartyl-tRNA Synthetase Has Glutamyl-tRNA Synthetase Activity

Genes ◽  
2019 ◽  
Vol 10 (4) ◽  
pp. 262 ◽  
Author(s):  
Udumbara M. Rathnayake ◽  
Tamara L. Hendrickson
Keyword(s):  
Amino Acid ◽  
Genetic Code ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Messenger Rnas ◽  
Indirect Pathway ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases

The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNAAsn via Asp-tRNAAsn. This intermediate is produced by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) that has retained its ability to also generate Asp-tRNAAsp. Here we report the discovery that ND-AspRS and its discriminating counterpart, AspRS, are also capable of specifically producing Glu-tRNAGlu, without producing misacylated tRNAs like Glu-tRNAAsn, Glu-tRNAAsp, or Asp-tRNAGlu, thus maintaining the fidelity of the genetic code. Consequently, bacterial AspRSs have glutamyl-tRNA synthetase-like activity that does not contaminate the proteome via amino acid misincorporation.

scholarly journals Properties and substrate specificities of the phenylalanyl-transfer-ribonucleic acid synthetases of Aesculus species

1970 ◽  
Vol 119 (4) ◽  
pp. 677-690 ◽  
Author(s):  
J. W. Anderson ◽  
L. Fowden
Keyword(s):  
Amino Acid ◽  
Maximum Velocity ◽  
Dienoic Acid ◽  
Trna Synthetase ◽  
Enoic Acid ◽  
Synthetase Activity ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Phenylbutyric Acid ◽  
Exchange Activity

1. Phenylalanyl-tRNA synthetases have been partially purified from cotyledons of seeds of Aesculus californica, which contains 2-amino-4-methylhex-4-enoic acid, and from four other species of Aesculus that do not contain this amino acid. The A. californica preparation was free from other aminoacyl-tRNA synthetases, and the contaminating synthetase activity in preparations from A. hippocastanum was decreased to acceptable limits by conducting assays of pyrophosphate exchange activity in 0.5m-potassium chloride. 2. The phenylalanyl-tRNA synthetase from each species activated 2-amino-4-methylhex-4-enoic acid with Km 30–40 times that for phenylalanine. The maximum velocity for 2-amino-4-methylhex-4-enoic acid was only 30% of that for phenylalanine with the A. californica enzyme, but the maximum velocities for the two substrates were identical for the other four species. 3. 2-Amino-4-methylhex-4-enoic acid was not found in the protein of A. californica, so discrimination against this amino acid probably occurs in the step of transfer to tRNA, though subcellular localization, or subsequent steps of protein synthesis could be involved. 4. Crotylglycine, methallylglycine, ethallylglycine, 2-aminohex-4,5-dienoic acid, 2-amino-5-methylhex-4-enoic acid, 2-amino-4-methylhex-4-enoic acid, β-(thien-2-yl)alanine, β-(pyrazol-1-yl)alanine, phenylserine and m-fluorophenylalanine were substrates for pyrophosphate exchange catalysed by the phenylalanyl-tRNA synthetases of A. californica or A. hippocastanum. Allylglycine, phenylglycine and 2-amino-4-phenylbutyric acid were inactive.

scholarly journals A Spectrophotometric Assay for Quantitative Measurement of Aminoacyl-tRNA Synthetase Activity

2012 ◽  
Vol 18 (4) ◽  
pp. 490-497 ◽  
Author(s):  
Igor Cestari ◽  
Kenneth Stuart
Keyword(s):  
Quantitative Measurement ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Aminoacyl Trna Synthetase ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Compound Screening ◽  
High Throughput Assay ◽  
Inhibition Studies ◽  
Trna Substrate

Aminoacyl-tRNA synthetases are enzymes that charge specific tRNAs with their cognate amino acids and play an essential role in the initial steps of protein synthesis. Because these enzymes are attractive targets for drug development in many microorganisms, there is a pressing need for assays suitable for compound screening. We developed (1) a high-throughput assay for measuring aminoacyl-tRNA synthetase activity and (2) an accompanying method for preparing the tRNA substrate. The assay can be performed in 96-well plates and relies on malachite green detection of pyrophosphate (Pi) as an indicator of aminoacyl-tRNA synthetase activity. Analysis of Trypanosoma brucei isoleucyl-tRNA synthetase (IleRS) activity showed that the assay exhibits sensitivity to picomoles of product and yielded a Z′ factor of 0.56. We show that this assay is applicable to other aminoacyl-tRNA synthetases and to enzyme inhibition studies. Using this assay, we found that the compound NSC616354 inhibits recombinant IleRS with an IC50 of 0.6 µM. Enzymology studies were also performed with rIleRS and its Km and kcat determined as 3.97 × 10−5 mol/L and 312 S−1, respectively. This assay will facilitate the screening of compounds to identify inhibitors of aminoacyl-tRNA synthetases.

scholarly journals Inhibitory mechanism of reveromycin A at the tRNA binding site of a class I synthetase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bingyi Chen ◽  
Siting Luo ◽  
Songxuan Zhang ◽  
Yingchen Ju ◽  
Qiong Gu ◽  
...  
Keyword(s):  
Binding Site ◽  
Catalytic Domain ◽  
Trna Synthetase ◽  
Rational Drug Design ◽  
Class I ◽  
Binding Assays ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Rational Drug ◽  
Trna Binding

AbstractThe polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNAIle binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNAIle. RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNAIle while binding synergistically with l-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.

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