Ribosomal subunits and ribosomal proteins of Tetrahymena thermophila

Abstract

Proteolytic degradation of ribosomal proteins occurs during the preparation of subunits of the cytoplasmic ribosomes of the protozoa Tetrahymenu thernioplrila and the isolated subunits are inactive. Addition of 5 mM iodoacetamide to cell suspensions before extraction inhibits proteolytic activity and permits isolation of active subunits. The protein complements of these subunits have been characterized in two different two-dimensional elcctrophoretic systems, and their molecular weights have been determined.Limited degradation of ribosomal proteins has been observed during isolation of ribosomes and ribosomal subunits from a variety of eukaryotic cells [l -61. This phenomenon was not observed in our earlier studies [7] with ribosomes of an amicronucleate Tetraliymeiza strain of unknown phenoset (Tetrahyniena 'pyr{forn~is' CGL) but has subscquently been shown to occur during preparation of ribosomes from several other amicronucleate strains [XI (and B. Petridou and F. Hayes, unpublished results). It also occurs, as shown here, during preparation of ribosomes from the micronucleate strain Tetruhymenu thermophila and leads to isolation of inactive ribosomal subunits unless protease inhibitors are present throughout the preparation process. Of a number of inhibitors tested, iodoacetamide was found to be the most effective. 40s and 60s ribosomal subunits prepared from T . thermophila in its presence are active in vitro and retain their activity during storage at low tcmperature. Alkylation of a small number of proteins in these subunits revealed by experiments with radioactive iodoacetamide appears not to impair their capacity to reassociate or to catalyse polyphenylalanine synthesis in vitro. The protein complements of active and inactive 40s and 60s ribosomal subunits of T. thernmphilu prepared in the presence and absence respectively of iodoacetamide have been characterized by polyacrylamide gcl electrophoresis in two different systems. The molecular weights of the proteins of active subunits have been estimated by SDS gcl electrophoresis.

Effect of heat shock on ribosome structure: appearance of a new ribosome-associated protein.

“…These changes, as indicated by the arrows, included an increase in the staining intensity of a doublet at 38 kDa, the disappearance of a band at 34 kDa, the disappearance of several minor bands at 30 kDa, and the appearance of a band at 22 kDa. When protease inhibitors were included in all steps of the ribosome isolation procedure, we found them to have no effect on these changes in protein arrays, indicating that they were not the result of proteolytic artifacts (27). Additional evidence, presented below, confirms that proteolysis could have played no role in generating these results.…”

Characterization of the phosphoproteins of Escherichia coli cells by electrophoretic analysis

“…For this, protein fractions were treated throughout by a mixture of two different protease inhibitors, iodoacetamide and PhMeS02F, active on sulfhydryl and hydroxyl groups of enzymes respectively [35]. The comparison of the phosphorylation patterns obtained with or without inhibitor treatment ( Fig.…”

“…95% (v/v) acetone at -2O"C, then centrifuged and dried under vacuum. In some experiments, fraction 530 was prepared in the presence of 1.3 mM phenylmethylsulfonyl fluoride (PhMeS02F) and 3 mM iodoacetamide [35].…”

Phosphorylation of a 40S ribosomal subunit protein in Tetrahymena

“…We have shown recently that the phosphorylation of S7, the putative Tetrahymena equivalent of S6 [14], is induced only if sodium chloride or high concentrations of Tris are present in the starvation buffer [lo]. Thus, in this organism, the process of phosphorylation could be uncoupled from that of starvation by manipulating the monovalent cationic constituents of the starvation buffers.…”