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.

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

“…Although the preparation of an appropriate pair of tRNA columns is much more rapid than the synthesis of a suitable aminoalkyladenylate analogue (see the preceding paper, Clarke & Knowles, 1977), there remain two disadvantages of tRNA columns. First, the lower resistance of tRNA columns to enzymic digestion necessitates at least a preliminary purification of the enzyme sample to remove ribonucleases.…”

“…Problems deriving from misrecognition are relatively easily eliminated with either type of ligand. In the preceding paper (Clarke & Knowles, 1977) we found that valyl-tRNA synthetase, which showed some retardation on a leucinyl-AMP column, was cleanly separated from the target enzyme by using either a two-column procedure or specific elution with substrate, and it seems reasonable to suppose that selective elution will invariably be possible in the few cases where misrecognition of a non-cognate aminoalkyladenylate occurs. Synthetase-tRNA misrecognition is more widespread, particularly in heterologous systems, although the problem can be minimized, if not eliminated, by the use of homologous tRNA.…”

“…In the present paper we report experiments carried out to establish the feasibility of the proposed method of preparation of the two affinity columns, and the use of such columns in the purification of valyl-tRNA synthetase from Bacillus stearothermophilus. Finally, the usefulness of tRNA columns is assessed in relation to the small-molecule systems discussed in the preceding paper (Clarke & Knowles, 1977). A preliminary report of some of this work has already appeared (Joyce & Knowles, 1974).…”