Aminoacyl, transfer sRNA ribosomes

Studies in this laboratory for the past several years have been concerned with the elucidation of the latter steps in this series of reactions. Specifically, efforts have been directed toward the characterization of the reaction involving the transfer of aminoacyl sRNA to mammalian ribonucleoprotein particles, the enzymatic and cofactor requirements, and possible intermediates in this process. The evidence obtained indicates that aminoacyl transfer is an enzymatic reaction requiring at least two enzyme fractions, which have been resolved and partially purified, GTP and a sulfhydryl compound further, the possibility exists that a ribosome-bound sRNA-amino acid (or peptide) compound is formed as an intermediate in this reaction. 

The transfer of amino acids from sRNA to protein was dependent on the concentration of ribosomes, aminoacyl sRNA, and transferring enzymes. Aminoacyl transfer was observed with less than 0.05 pmole of GTP per ml., and the reaction exhibited a sulfhydryl requirement which was met by a variety of compounds. Transfer of several sRNA-bound C14-amino acids to ribosomal protein was observed with the puri-ified transferases I and n. 

Incubations of crude pH 5 Supernatant with several C14-aminoacyl sRNA preparations, differing only in the nature of the C14-amino acid, showed that all amino acids tested were incorporated into ribosomal protein. With combined transferases I and n, the results presented in Table VII indicated that all of the amino acids tested were also incorporated in the presence of these purified fractions (8). When either of the transferases was omitted from these incubations, little amino acid transfer was observed with any of the C14-aminoacyl sRNA preparations. Variations in total amounts of C14 incorporated, as shown here, are probably due to variations in the specific radioactivity of the various sRNA-bound amino acids used. These purified transferase preparations did not catalyze the incorporation of free amino acids into sRNA or ribosomes. 

The transfer of labeled amino acids from aminoacyl sRNA to purified rat-liver ribonucleoprotein particles has been shown to require GTP, and a soluble portion (pH 5 Supernatant) of the cell. An enzyme fraction, aminoacyl transferase (or polymerase) I, purified from the pH 5 Supernatant was found to catalyze the transfer of amino acid to protein with microsomes, but not with the more purified ribonucleoprotein particles (ribosomes). When transferase I was supplemented with glutathione and a microsomal extract, microsomal aminoacyl transferase (or polymerase) H, transferring activity was restored. Since the pH 5 Supernatant was active in catalyzing the transfer of amino acids from sRNA to ribosomal protein, it was concluded that both transferring activities were present in this crude fraction. Resolution of the two activities from the pH 5 Supernatant fraction was obtained by salt-fractionation procedures. Neither enzyme fraction was active when incubated individually or with glutathione, but together in the presence of... 

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