The production of biologically active subparticles from rabbit reticulocyte ribosomes

Abstract

The effect of exposing rabbit reticulocyte ribosomes to concentrated solutions of potassium chloride was examined by: (a) dialysis against 0·5m-potassium chloride; (b) zone centrifugation through a sucrose gradient in 0·5m-potassium chloride; (c) differential centrifugation of a solution made 0·5m with respect to potassium chloride. The products of each treatment and their ability to support protein synthesis in a reticulocyte cell-free system, in the presence and in the absence of polyuridylic acid, were examined. The following results were found. (1) Exposing the polysomes to 0·5m-potassium chloride was not a sufficient condition for the complete dissociation of ribosomes into subparticles; the reaction showed a concentration-dependence, implying the existence of an equilibrium between the various ribosomal species. Disturbance of the equilibrium by removing certain products, as in zone centrifuging, can lead to complete dissociation. (2) The subparticles produced by dialysis or sucrose-gradient fractionation were biologically inactive and unstable. (3) The pellet obtained by differential centrifuging consisted of subparticles, if suspended in a Mg2+-free buffer; addition of Mg2+ converted about 30% of the material into heavier sedimenting species, and the preparation had 20–40% of the activity of the untreated control polysomes in the cell-free system. Addition of the 0·5m-potassium chloride supernatant fraction resulted in further apparent reconstitution of sub-particles into ribosomes and polysomes and in a 50–100% restoration of biological activity. When both polyuridylic acid and supernatant factors were present incorporations similar to or higher than those of the control were attained.