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Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.

ABSTRACT: The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed.

SUBMITTER: Clarke CM

PROVIDER: S-EPMC1183672 | biostudies-other | 1977 Nov

REPOSITORIES: biostudies-other

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Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

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| S-EPMC1183673 | biostudies-other

The extraction and assay of aminoacyl-transfer-ribonucleic acid synthetases of tobacco leaf.

Project description:1. Aminoacyl-transfer-RNA synthetase activity in extracts prepared from tobacco leaf was increased 3-5-fold when sodium thioglycollate (30mm) and magnesium chloride (16mm) were included in the extraction medium. Omitting sucrose (0.45m) from the extraction medium did not alter the activity. 2. Activity was a linear function of enzyme concentration up to 1 disk (30mg. fresh wt.)/ml. and was not affected by dialysis at any concentration. 3. Activity increased about 13-fold above control values when a mixture of 21 amino acids and amides (1mm) was added to the reaction mixture. 4. Under the conditions used in the standard assay for aminoacyl-transfer-RNA synthetase activity K(m) (ATP) was 0.65mm and K(m) (l-amino acids) was 70mum. 5. Activity above the control value was found with all amino acids and amides tested except alanine, arginine, glutamic acid, glutamine and hydroxyproline. Activity was highest with leucine, isoleucine, valine, cysteine and histidine. Total activity with a mixture of 21 amino acids and amides was 20% lower than the total activity of the enzymes assayed separately.

| S-EPMC1270057 | biostudies-other

Myocardial aminoacyl-transfer-ribonucleic acid synthetase and aminoacyl-transferring enzyme activity.

Project description:The properties of cytoplasmic aminoacyl-tRNA synthetase and aminoacyl-transferring enzymes in the myocardium were examined and methods for the assay of the activity of these enzyme systems were developed. Aminoacyl-tRNA synthetase activity was measured from the rate of incorporation of (14)C-labelled amino acid into aminoacyl-tRNA. Transferase activity was measured from the rate of incorporation of amino[(14)C]acyl-tRNA into protein in the presence of a standard preparation of hepatic ribosomes. Aminoacyl-tRNA synthetase activity is labile once the heart has been homogenized, whereas transferase activity is stable. The source of energy for synthetase activity is ATP; that for transferase is GTP. Transferase activity was inhibited by puromycin and stimulated by dithiothreitol, whereas synthetase activity was unaffected.

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Adenosine triphosphate consumption by bacterial arginyl-transfer ribonucleic acid synthetases.

Project description:ATP consumption by arginyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus has been investigated by the firefly luciferin--luciferase assay. Arginyl-tRNA synthetase from E. coli utilizes ATP only for aminocylation of tRNA with a 1:1 stoicheiometry. In contrast, we have shown an adenosine triphosphatase activity of arginyl-tRNA synthetase from B. stearothermophilus in the absence of tRNAArg. Dowex chromatography revealed the formation of ADP by the thermophile enzyme; under aminoacylation conditions, AMP was also formed in amounts stoicheiometric with arginyl-tRNA formation.

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Activity of aminoacyl-transfer-ribonucleic acid synthetases in tobacco-leaf tissue in relation to senescence and to the action of 6-furfurylaminopurine.

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Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. Formation and properties of an aminoacyl-transfer ribonucleic acid-transferase I complex.

Project description:1. Transferase I of rat liver binds aminoacyl-tRNA to form a relatively stable complex, which is retained on cellulose nitrate filters. This reaction proceeds at both 0 degrees C and 37 degrees C and is inhibited by GTP. The resulting product is stabilized by GTP and Mg(2+). 2. Only very low quantities of deacylated tRNA are bound by transferase I. 3. Methods are described for the preparative isolation of the transferase I-aminoacyl-tRNA complex from incubation mixtures by using ion-exchange procedures. 4. The transferase I-aminoacyl-tRNA complex becomes readily bound to ribosomes. The presence of Mg(2+) is essential for the binding. GTP stimulates this reaction but is not absolutely required. 5. It is concluded that the formation of the transferase I-aminoacyl-tRNA complex may be the primary reaction in the binding of aminoacyl-tRNA to mammalian ribosomes and that, unlike in bacterial systems, GTP is not absolutely required for this step.

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Horizontal transfer of drug-resistant aminoacyl-transfer-RNA synthetases of anthrax and Gram-positive pathogens.

Project description:The screening of new antibiotics against several bacterial strains often reveals unexpected occurrences of natural drug resistance. Two examples of this involve specific inhibitors of Staphylococcus aureus isoleucyl-transfer-RNA synthetase 1 (IleRS1) and, more recently, Streptococcus pneumoniae methionyl-tRNA synthetase 1 (MetRS1). In both cases, resistance is due to the presence of a second gene that encodes another synthetase (IleRS2 or MetRS2). Here, we show that both S. pneumoniae MetRS2 and S. aureus IleRS2 have closely related homologues in the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax. Furthermore, similar to drug-resistant pathogens, strains of B. anthracis and its closest relative, B. cereus, also have wild-type ileS1 and metS1 genes. Clostridium perfringens, the causative agent of gangrene, also has two metS genes, whereas Oceanobacillus iheyensis isolated from deep-sea sediments has a single ileS2-type gene. This study shows the importance of understanding complex evolutionary networks of ancient horizontal gene transfer for the development of novel antibiotics.

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Affinity chromatography of thiol-containing purines and ribonucleic acid.

Project description:Evidence is presented for the specific interaction of 6-mercaptopurine with mercurated cellulose. Following from this, a new method is described for the affinity chromatography of thiol-containing molecules and of RNA containing incorporated 6-thioguanosine on columns of mercurated cellulose. This technique may find application in the study of RNA metabolism and gene expression.

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Non-canonical Amino Acid Substrates of E. coli Aminoacyl-tRNA Synthetases.

Project description:In this comprehensive review, I focus on the twenty E. coli aminoacyl-tRNA synthetases and their ability to charge non-canonical amino acids (ncAAs) onto tRNAs. The promiscuity of these enzymes has been harnessed for diverse applications including understanding and engineering of protein function, creation of organisms with an expanded genetic code, and the synthesis of diverse peptide libraries for drug discovery. The review catalogues the structures of all known ncAA substrates for each of the 20 E. coli aminoacyl-tRNA synthetases, including ncAA substrates for engineered versions of these enzymes. Drawing from the structures in the list, I highlight trends and novel opportunities for further exploitation of these ncAAs in the engineering of protein function, synthetic biology, and in drug discovery.

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

Project description: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.

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