| Description |
Monomeric ribosomal particles were isolated from canine pancreas. These particles were active in Poly U-directed polyphenylalanine synthesis. They contained 53% protein. From these SOS monomers, 40S and 60S ribosomal subunits were isolated using a hyperbolic sucrose gradient. The small subunit preparation was shown to be free of contamination by the large subunit after this procedure. The large subunit required additional purification. Both large and small subunits, after purification, had no independent activity in phenylalanine incorporation but activity was quite satisfactory when the preparations were combined. The subunits were characterized with respect to sedimentation coefficient and protein content. Magnesium, monovalent cation, and ribosome concentration optima were identified in the phenylalanine incorporation assay. Ribosomal small subunits from one preparation were used to survey the effects of the entire range of potassium chioride concentrations. When combined with reference 60S particles, the treated 40S subunits shov/ed no significant change in phenylalanine-incorporatlng activity until exposure to 0.95 M KCl. After treatment with 0.95 M KCl, the 40S subunits retained 80% of the phenylalanine-incorporating activity of the 0.40 M KCl-treated subunits (the lowest salt treatment used in the study). After treatment v/ith 1 .25 M KCl, the subunits had cniy 15-25% of the phenylalanine-incorporating activity of the 0.40 M KCI-treated 40S subunits. Concomitant decreases in the sedimentation coefficient and protein content of the subunits were also observed. Attempts to separate dissociated ribosomal protein and the remaining core particles treated with 1 .25 M KCl by the centrifugation of the salt-treated particles through a 40% sucrose cushion led to the observation that ribosomal subparticles isolated in this manner retained full phenylalanine-incorporating activity. Other isolation techniques had been previously observed to result in inactivated particles. Experiments were performed to elucidate the mechanism by which the 40% sucrose cushion was stabilizing the high-salt-treated 40S subunits. Some of the activity lost in the isolation of the subunits without the 40% sucrose cushion could be recovered by reisolating the particles on a sucrose cushion, but this reactivation was not dependent on any material which had been previously removed by the salt treatment. This seemed to rule out a reconstitution-type process. Experiments were also conducted to determine the composition of the salt-treatment, dissociation mixture. Two-dimensional gel-electrophoresis of the proteins of the various particles isolated in the study was performed and the results are presented. Most of the active 40S subparticles contained 29-31 protein spots while inactive preparations contained fewer proteins. However, one active preparation contained only 23 possible protein spots which is fewer than previously reported for a small subunit preparation from a eukaryotic system. Finally, the possible mode of action of the sucrose cushion is discussed wil-h emphasis on the possibility of a conformational change occurring in the subunits during centrifugation. |
