Studies on the synthesis and turnover of arginine and leucine: tRNA ligases in cultured tobacco cells

Abstract

A technique was developed for assaying amino acid: tRNA ligases extracted from tobacco XD cells grown in chemically defined medium (M-1D). The technique was based on the physiological enzymic reaction in which amino acid is aminoacylated to tRNA. tRNA was obtained from tobacco XD cells using a phenol extraction procedure. For two enzymes, arginine: tRNA ligase and leucine: tRNA ligase, assay conditions were optimised. Both enzymes had similar Km values for their cognate amino acids; were found to be unstable when stored at -10 and their activity was inhibited by ammonium sulphate and caesium chloride. During growth of tobacco XD cells, these two enzymes increased in activity. Amino acids appeared not to be involved in their regulation and attempts to perturb in vivo levels of aminoacyl-tRNA by use of amino acid analogues were unsuccessful. The use of the density labelling technique, which allows a distinction between pre-existing enzyme molecules and those that are newly synthesised, indicated that in M-ID both arginine; and leucine: tRNA ligases were synthesised de novo. Leucine: tRNA ligase was also degraded and therefore turned over as it increased in activity. The density labelling data did not allow a similar conclusion for arginine: tRNA ligase. During cell growth in nitrateless M-1D, there was no increase in the activity of arginine: and leucine: tRNA ligases, but both anzymes were found to be synthesised de novo. It was concluded, therefore, that they were both degraded and so turned over in nitrateless M-ID. Arginine: and leucine: tRNA ligases appeared to be synthesised from different amino acid pre-cursor pools and DEAE cellulose chromatography of enzyme extracts revealed the presence of three ligase species cognate for arginine but only two species cognate for leucine. The species cognate for arginine were in approximately equal proportions whereas one of the species cognate for leucine accounted for 80% of the total enzyme activity. The possibility that these multiple enzymic species might be responsible for the inability to demonstrate degradation of the arginine anzyme in M-1D was discussed. An accurate determination of the turnover rates of these/ these two enzymes could not be obtained due to the effects of re-cycling of total cell protein, but a comparison of turnover rates was attempted. The possible mode of regulation of these enzymes was discussed in relation to our observations and to those found in other systems.