Protein synthesis by cell-free extracts of tobacco leaves: III. Comparison of the physical properties and protein synthesizing activities of 70 s chloroplast and 80 s cytoplasmic ribosomes

Abstract

Young tobacco leaves contain two classes of ribosomes in about equal quantities. From ultracentrifugal analyses of chloroplast and cytoplasmic extracts, it is concluded that the 70 s class of ribosomes is located in the chloroplasts and the 80 s class in the cytoplasm. The 70 s ribosomes in a chloroplast extract are 10 to 20-fold more active in protein synthesis (200 to 300 μμmole [14C]valine incorporated/mg ribosome/30 minutes) than the 80 s ribosomes in a cytoplasmic extract. If the chloroplasts are disrupted by homogenization of leaves in a medium of low molarity, the 70 s ribosomes become mixed with the cytoplasm and are less active than the 70 s ribosomes in a chloroplast extract. Inactivation does not seem to result from nuclease action, or deficiencies in sRNA and/or activating enzymes or messenger RNA. Pelleting the ribosomes of a chloroplast extract markedly reduces their incorporating activity. In contrast, the 80 s ribosomes of a cytoplasmic extract are more active after purification. The 70 s and 80 s ribosomes have different magnesium ion requirements for maximum incorporating activity, the optimum concentration being 11 to 15 mM for 70 s ribosomes and 5 ml for 80 S ribosomes. At low concentrations of magnesium ions, most of the 70 s ribosomes dissociate into subunits of 50 s and 35 s, which reconstitute 70 s ribosomes on restoration of the magnesium. The 80 s ribosomes are resistant to dissociation in low concentrations of magnesium ions, but on prolonged dialysis dissociate into 58 s and 35 s particles, which do not reconstitute 80 s particles. 80 s ribosomes aggregate preferentially at high concentration of magnesium. The corrected sedimentation coefficients (S20.w0) of the 70 s and 80 s ribosomes are 69·9 and 82·0s, respectively.