The purification and properties of the alanyl-transfer ribonucleic acid synthetase of tomato roots

Abstract

The alanyl-s-RNA synthetase of tomato roots has been purified by ammonium sulphate precipitation, adsorption on calcium phosphate gel and DEAE-cellulose chromatography and its properties have been investigated. Enzyme activity was measured by using the hydroxamate assay, the [P32]pyrophosphate-ATP-exchange assay and the [C14]alanyl-s-RNA assay. The purified enzyme was specific for L-alanine and was activated by Mg2+ions and to a smaller extent by CO2+and Mn2+ ions. It was free from adenosine triphosphatase, pyrophosphatase and ribonuclease and possessed a specific activity comparable with that of the most highly purified aminoacyl-s-RNA synthetases from animal and microbial systems. The properties of the purified enzyme were similar in many respects to most other highly purified aminoacyl-s-RNA synthetases. It differed, however, in that the pH optimum of the hydroxamate assay was almost the same as that of the pyrophosphate-ATP-exchange assay and in requiring a high concentration of L-alanine for maximum activity (100 [mu]moles/ml.). The purified enzyme was not absolutely specific for tomato-root s-RNA; slight activity was also observed with yeast s-RNS. The properties of this enzyme are fully consistent with the suggestion that the enzymic formation of alanyl-s-RNA proceeds via the intermediate formation of alanyl acyl-adenylate with the elimination of pyrophosphate from ATP. It remains to be shown the extent to which alanyl-s-RNA participates further in subsequent stages of protein synthesis in plants.