ISOLATION OF POLYRIBOSOMES AND CHARACTERISTICS OF AN AMINO ACID-INCORPORATION SYSTEM FROM LIVER OF A PRIMATE (PAPIO PAPIO)

1. 1. A procedure is described for the isolation of a polyribosomal preparation from baboon liver (Papio papio). Sucrose-gradient centrifugation shows this polysomal population to consist primarily of aggregates containing three or more ribosomes. The yield of polyribosomes and their activity as assayed in a cell-free system is shown to be dependent upon the method of homogenization (pestle clearance of 0.9 mm) and the concentration of deoxycholate (0.50%). 2. 2. Under optimal conditions of isolation the polyribosomal preparation is able to support the incorporation of [14C]valine into protein when supplied with the appropriate cofactors. The cell-free system shows a strong dependence for polyribosomes, ATP, and an ATP-generating system, and also requires GTP, a pH-5 enzyme fraction and Mg2+ for maximal activity. The dependency upon GTP is greater than that reported for other cell-free systems. The optimal Mg2+ concentration is 6-7 mM, and the activity of the cell-free system is not dependent upon added K+. 3. 3. Incorporation is inhibited by incubation with ribonuclease or NaF which dissociate polyribosomes into monomeric forms. Inhibition is also caused by the addition of puromycin and to a lesser degree by similar concentrations of cycloheximide. From the data concerning the inhibition by NaF, it is suggested that the incorporation of amino acids into peptides represents both chain initiation and the completion of previously existing chains. © 1969.