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Factors controlling aminoacyl-transfer-ribonucleic acid synthesis in vitro by a plant system.

ABSTRACT: 1. Factors affecting aminoacyl-tRNA synthesis in vitro by cell-free preparations from bean leaves were investigated. 2. Evidence was obtained that optimum concentrations as well as correct ratios of Mg(2+) and ATP are required for aminoacyl-tRNA synthesis in the bean-leaf system. 3. The results indicated that pH is a controlling factor having differential effects on the formation of individual aminoacyl-tRNA species. The possible micro-regulatory function of pH in protein synthesis in vivo is discussed with special reference to alanyl-tRNA formation. 4. Very low rates of alanine-stimulated pyrophosphate exchange were observed in the absence of tRNA. This observation is discussed relative to proposals about the mechanism of aminoacyl-tRNA synthesis.

SUBMITTER: Tao KL

PROVIDER: S-EPMC1176597 | biostudies-other | 1971 Feb

REPOSITORIES: biostudies-other

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Project description:An acute administration of phenylalanine to neonatal animals has been reported to result in large decreases in the intracellular concentrations of several essential amino acids in neural tissue, as well as an inhibition of neural protein synthesis. The present report evaluates the effects of the loss of amino acids on the concentrations of aminoacyl-tRNA in vivo, with the view that an alteration in the concentrations of specific aminoacyl-tRNA molecules could be the rate-limiting step in brain protein metabolism during hyperphenylalaninaemia. tRNA was isolated from saline- and phenylalanine-injected mice 30-45 min after injection, by using a procedure designed to maintain the concentrations of aminoacyl-tRNA present in vivo. Periodate oxidation of the non-acylated tRNA and aminoacylation with radioactively labelled amino acids was used to determine the proportion of tRNA that was present in vivo as aminoacyl-tRNA. Although decreases in the intracellular concentrations of alanine, lysine and leucine were observed after phenylalanine administration, the concentrations of alanyl-tRNA, lysyl-tRNA and leucyl-tRNA actually increased by 15%. Although tryptophan has been suggested to be rate-limiting during hyperphenylalaninaemia, the proportion of tryptophan tRNA that was acylated was maximal in both normal and hyperphenylalaninaemic animals. This unexpected increase in aminoacyl-tRNA concentration is discussed as perhaps a secondary effect resulting from the phenylalanine-induced inhibition of protein synthesis. In contrast, the proportion of methionine tRNA that was acylated in vivo after phenylalanine administration was demonstrated to be decreased by approx. 17%. When the isoaccepting species of methionine tRNA were separated by reverse-phase column chromatography, three species were separated, one of which was demonstrated to be the initiator species, tRNAfMet, by the selective aminoacylation and formylation with Escherichia coli enzymes. After the administration of phenylalanine, the acylation of each of the three methionine tRNA species was decreased, with the initiator species being lowered by 10%. This effect on aminoacylation of tRNAfMet may be the primary step by which phenylalanine affects neural protein synthesis, and this is consistent with previous reports that re-initiation may be inhibited during hyperphenylalaninaemia.

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Factors controlling aminoacyl-transfer-ribonucleic acid synthesis in vitro by a plant system.

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