Studies on the small subunit of rat liver ribosomes: Some biochemical properties with specific references to the reconstruction of the small subunit

Abstract

1. The chemical analysis and CsCl buoyant density gradient centrifugation showed that the protein content of the 32-S ribosomal subunit, prepared from rat liver ribosomes by EDTA treatment, was lower than that of the active 40-S subunit, prepared by incubation with a high concentration of KCl. The same analyses of the EDTA-treated 47-S subunit and the active 60-S subunit showed similar protein contents between two types of ribosomal large subunits. The EDTA-treated 32-S subunit prepared in the presence of the liver ribonuclease inhibitor showed poly(U)-binding activity comparable to the active 40-S subunit, although the activity of poly(U)-dependent polyphenylalanine synthesis was very low. 2. Protein-depleted particles (40-S core particles) and split proteins were prepared from the active 40-S subunit by treatment with 0.4 M LiCl in the presence of 5 mM MgCl 2. About 25 % of protein was dissociated from the active 40-S subunit by this treatment. The selective dissociation of proteins from 40-S subunit was shown by acrylamide gel electrophoresis of split proteins and 40-S core particles. The reconstructed particles from core particles and split proteins from the active 40-S subunit showed definite activity in poly(U)-dependent polyphenylalanine synthesis in conjunction with the active 60-S ribosomal subunit. 3. By incubation of 18-S ribosomal RNA with proteins of the 40-S ribosomal subunit, particles with s value of about 32 S were reconstructed. The protein content of the 32-S reconstructed particles was somewhat lower than that of the EDTA-treated 32-S subunit and acrylamide gel electrophoresis of the proteins of the particles showed a complete set of protein components of the original 40-S subunit, although the densitogram showed a difference in some bands between the 40-S subunit and the reconstructed particles. The reconstructed 32-S particles showed definite activity in poly(U)-binding, but their activity in poly(U)-dependent poly-phenylalanine synthesis in conjunction with the active 60-S sbunit was very low.