Isolation of Active Ribosomal Subunits from Yeast

We have designed a method for the large scale isolation of pure and highly active subunits from yeast cytoplasmic ribosomes. The method consists of the incubation of yeast protoplasts in the presence of 1 mM NaN3. This causes the disappearence of polysomes and the formation of 80‐S ribosomes that can be dissociated in 0.5 M KCl. In this way over 90% of the ribosomes can be dissociated into subunits. The subunits are isolated by centrifugation in a B‐XV zonal rotor using an equivolumetric sucrose gradient. When this separation is performed at 5°C, dimerization of 40‐S particles to 65‐S particles occurs and no pure subunits can be obtained. At 10°C, there is no dimerization and the 60‐S + 40‐S subunits obtained are less than 1% cross‐contaminated. In poly(U)‐directed protein synthesis one subunit pair polymerizes phenylalanine residues at a rate of 1.7 residue per min. About 70% of the subunits were estimated to participate in this synthesis. Subunits prepared in the absence of dithiothreitol still bound phenylalanyl‐tRNA, but were inactive in phenylalanine polymerization. The 60‐S subunits prepared without dithiothreitol were as active as those prepared in the presence of dithiothreitol when their peptidyltransferase activity was tested.