Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Attwood MM

PROVIDER: S-EPMC1207195 | biostudies-other | 1965 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets