ISOLATION OF LABELLED AMINOACYL TRANSFER RNA FROM MUSCLE - STUDIES OF ENTRY OF LABELLED AMINO ACIDS INTO ACYL TRANSFER RNA LINKAGE IN SITU AND ITS CONTROL BY INSULIN

Methods have been investigated for measurement of the specific activity of labelled amino acids contained in aminoacyl-tRNA of muscle after incubation of diaphragm or perfusion of heart with labelled amino acids. Extraction of tissue homogenates with hot trichloroacetic acid, alkali or hot NaCl solution were all found to solubilise sufficient protein to make measurement of radioactivity specifically derived from aminoacyl-tRNA impossible. Treatment of the pH 5 precipitate or 100 000×g supernatant with phenol and/or bentonite resulted in reasonable purification of tRNA as judged by density gradient profiles. Two extractions with bentonite at pH6 without phenol was found to be a satisfactory compromise in terms of yield, purity of product and ease of manipulation. By such means aminoacyl-tRNA labelled with tyrosine and leucine was satisfactorily isolated, but the correlation of radioactivity with absorbance during gradient analysis was poor when glycine, proline and methionine were the source of label. The specific activity calculated for the amino acid of the aminoacyl-tRNA appeared in most cases to reach values close to that of the free amino acid added to the system. Turnover of the aminoacyl group was rapid. Labelling of the leucyl- and tyrosyl-tRNA was raised in the presence of insulin. © 1969.