Structure and function of mammalian ribosomes. I. Isolation and characterization of active liver ribosomal subunits.

Wo have developed a method for preparation and purification of active ribosomal subunits from rat and mouse liver. Our technique involvea incubating purified polysomes with all components for protein synthesis until all competent ribosomes have terminated and released their polypeptide chain. Upon kreatment of such an incubation mixture with 0.5 M-KCl, followed by sucrose gradient centrifugation in 0.3 M-KC1 and 0.002 or 0.003 M-magnesium acetate, 80 to 90% of all ribosomea are found as 60 s and 40 s subunits. These subunits can be concentrated from the gradient fractions by ethanol precipitation. The purified subunits, when recombined, spontaneously associate to 80 s couples in the absence of transfer RNA, messenger RNA or supernatant factors. When supplied with poly U and the other components required for in, w&o polypeptide synthesis, the subunits polymerize 16 to 20 phenylalanine residues per subunit couple present in the reaction mixture. Furthermore, at least 50 to 65% of the subunits actually participate in polyphenylalanine synthesis. At least 90% of the RNA of the purified subunits is intact as shown by sedimentation analysis of lithium dodecyl sulfatetreated particles. The purified 60 s subunit contains the enzymic site for the catalysis of peptide bond formation. This reaction is sensitive to anisomycin, an inhibitor of mammalian protein synthesis, but is not affected by the bacterial inhibitor chloramphenicol nor by cyclohexamide, an inhibitor of translocation in mammalian systems. The proteins from 40 s and 60 s subunits and from polysomes were analyzed and compared by acrylamide gel okkrophorosis.