Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.
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.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D007930:Leucine; D008024:Ligands; D012685:Sepharose
D008024:Ligands; D000249:Adenosine Monophosphate; D012685:Sepharose; D000604:Amino Acyl-tRNA Synthetases; D007930:Leucine