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.