The purification and properties of the alanyl-transfer ribonucleic acid synthetase of tomato roots.
1. 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. 2. Enzyme activity was measured by using the hydroxamate assay, the [(32)P]pyrophosphate-ATP-exchange assay and the [(14)C]alanyl-s-RNA assay. The purified enzyme was specific for l-alanine and was activated by Mg(2+) ions and to a smaller extent by Co(2+) and Mn(2+) 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. 3. 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 (100mumoles/ml.). 4. The purified enzyme was not absolutely specific for tomato-root s-RNA; slight activity was also observed with yeast s-RNA. 5. 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.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine; D011756:Diphosphates / Q000378:metabolism; D005638:Fruit; D006863:Hydrogen-Ion Concentration; D006898:Hydroxylamines / Q000378:metabolism; D066298:In Vitro Techniques; D008025:Ligases / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D010945:Plants, Edible / Q000201:enzymology*; D012343:RNA, Transfer
D011756:Diphosphates; D006898:Hydroxylamines; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D008025:Ligases; D008274:Magnesium; D000409:Alanine