Controlled dissociation of rabbit reticulocyte ribosomes and its effect on hemoglobin synthesis

Abstract

The state of aggregation of ribosomes can be ascertained by analytical ultra-centrifugation. The extent of aggregation of ribosomes, including those from rabbit reticulocytes, depends on the Mg 2+ concentration and ionic strength of the medium. In the usual incubation medium in which rabbit reticulocyte ribosomes incorporate amino acids into hemoglobin, the ribosomes exist predominantly as 70 s particles. In solutions without Mg 2+ , or with low Mg 2+ concentrations, one 70 s ribosome reversibly dissociates into one 50 s and one 30 s particle. Various methods are available for the removal of Mg 2+ from reticulocyte ribosomes in order to achieve dissociation, among them pyrophosphorolysis. By varying the relative concentration of ribosomes and pyrophosphate, any desired extent of dissociation into subunits is feasible. Ribosomes were tested for their protein synthetic capacity after reversible dissociation. It was observed that once ribosomes had been completely dissociated into subunits they lost the capacity to incorporate amino acids into protein and none of the usual supernatant factors was capable of restoring this property. The experiments present evidence that in the usual preparation of reticulocyte ribosomes two classes of particles are actively participating in protein synthesis; a small portion—about 5%—of the ribosomes is 10 times more active, and more resistant to dissociation, than the remaining 95% of the ribosomal population.