Precipitation of Saccharomyces cerevisiae ribosomes by ethanol under experimental conditions that do not release the ribosomal proteins can affect the activity of the particles. In the presence of 0.4 M NH4Cl and 50% ethanol only the most acidic proteins from yeast and rat liver ribosomes are released. At 1 M NH4Cl two more non-acidic proteins are lost from the ribosomes. The release of the acidic proteins causes a small inactivation of the polymerizing activity of the particles, additional to that caused by the precipitation itself. The elongation-factor-2-dependent GTP hydrolysis of the ribosomes is, however, more affected by the loss of acidic proteins. These proteins can stimulate the GTPase but not the polymerising activity when added back to the treated particles. Eukaryotic proteins cannot be sustituted for bacterial acidic proteins L7 and L12. We have not detected immunological cross-reaction between acidic proteins from Escherichia coli and those from yeast, Artemia salina and rat liver or between acidic proteins from these eukaryotic ribosomes among themselves.The existence in eukaryotic ribosomes of acidic proteins with electrophoretic mobility and molecular weight similar to that of proteins L7 and L12 from Escherichia coli ribosomes has been reported [l-41. In some cases antibodies against the bacterial proteins were reported to cross-react with the eukaryotic proteins [I].Functional similarities between acidic proteins from bacterial and higher cells have been reported [2,6] using particles deprived of these proteins by washing the ribosomes with 1 M NH4Cl and 50% ethanol as described for E. coli [7]. It was later found that this treatment induces the release of two proteins in addition to the acidic ones [3]. Therefore, the conclusions drawn from this type of experiment must be re-examined.We have found experimental conditions that permit the selective release of most acidic proteins from rat liver and yeast ribosomes. The roles of the acidic proteins in both types of ribosomes have been investigated using these particles. The results presented in this report indicate differences in the requirements of acidic proteins for the function of eukaryotic and prokaryotic ribosomes.Ahhreviatiun. EF-1, 2, etc, elongation factors 1, 2, etc MATERIALS AND METHODS Preparation of RibosomesSaccharornyces cerevisiae, strain Y 166 was grown in yeast extract/peptone/glucose medium [S] in a LabLine/S.M.S. Hi-density fermentor, up, to late exponential phase. Cells were broken by sea-sand grinding and the ribosomes were washed in high-salt buffers [9] and finally resuspended in 80 mM KCI, 10 mM MgCL and 20 mM Tris-HCI, pH 7.4 (buffer 1). The particles were stored in liquid nitrogen. Rat liver ribosomes were prepared as described elsewhere [lo]. Preparation of Core Particles and Split Protein FractionsThe ribosomes were dissolved at 4 mg/ml in 10 mM imidazol-HC1 buffer pH 7.4, containing 20 mM MgC12, 1 mM 2-mercaptoethanol and either 0.4 M or 1 M NH4Cl. Ethanol was added up to 50% (v/v) final concentration in two steps...

Protein-deficient particles have been obtained by treating rat liver 80-S ribosomes or their 60-S subunits with 1 M NH4Cl in the presence of 50 % ethanol at 0 "C (Po-cores) and 37 "C (P37-cores).The Po-cores from 8 0 4 ribosomes are totally inactive in polyphenylalanine synthesis but fully active in the 'fragment assay' to test peptidyl transferase activity. The polymerizing activity of the cores is restored up to 40-50% of control activity by incubation in the presence of the split proteins. Three proteins are totally lost in the treatment, namely proteins L12, L40/41 and S25. A series of up to nine different spots in the region of the L40/41 proteins are detected when the split fraction is analyzed by two-dimensional electrophoresis. This series of spots is, however, reduced to only two proteins when the second dimension is carried out in the presence of sodium dodecylsulphate.80-S ribosome-derived P37-cores are about 80 % active in the 'fragment reaction' while 60-Ssubunit-derived particles are inactive in this assay. The inhibitory effect of a number of antibiotics is differentially affected by the treatment suggesting different localization of their binding sites. A comparative study of the proteins released by treatment in the two types of particles suggests the involvement in the peptidyl transferase center of the ribosome of one or more of the following proteins: L21, L24, L27, L28-and L36.

Rat liver ribosome treatment with ethanol and 1 M NH4Cl releases some 31-33 ribosomal proteins. This split protein fraction binds Phe-tRNA, Ac-Phe-tRNA, Met-tRNAM and f-Met-tRNAF in the absence of K+ and Mg++ ions. When the split protein fraction is passed through Sephadex G-100 only six proteins are retained in the column: S10, S14, S15, S19, L35, and L36. The aminoacyl-tRNA binding activity of this protein fraction retained in the Sephadex G-100 column is similar to that of the total split protein fraction, suggesting that the above six proteins, or only some of them, are involved in the binding reaction.