The dissociation of Yoshida hepatoma ribosomes into active particles

1. The sedimentation pattern of Yoshida hepatoma ribosomes shows mainly a high dimer peak and an intermediate peak between those of monomer and dimer. 2. The treatment of the postmitochondrial supernatant with EDTA or potassium chloride leads to the dissociation of ribosomes into subunits, through a decrease ofthe ribosomal Mg²⁺/phosphorus ratio. Provided that the deprivation of endogenous Mg²⁺ is not complete, the subunits reassociate into active polyribosomes after addition of magnesium chloride to the medium. If the Mg²⁺/phosphorus ratio is lowered below 0.01 (μmol/μmol), structural changes occur, that become evident by a loss of protein and by a decreased sedimentation rate, which render the subribosomal particles unable to reassociate. 3. In the re-formation of polyribosomes from subunits, during the progressive increase of the concentration of magnesium chloride in the medium, the formation of monomeric ribosomes seems to be intermediate. 4. A dimerization of monomers and subunits occurs at concentrations of magnesium chloride greater than those required for the re-formation of polyribosomes and a preincubation for 1h at 0°C is necessary for the maximum dimerization, whereas the complete reconstitution of polyribosomes is immediate.     

The production of biologically active subparticles from rabbit reticulocyte ribosomes

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 Mg(2+)-free buffer; addition of Mg(2+) 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.     

A simple general method to determine the proportion of active ribosomes in eukaryotic cells

The resistance of 80S ribosomes derived from active polyribosomes to dissociation in high KCl cone, can conveniently be used to assay the proportion of active ribosomes in eukaryotic cells. The method described has been useful in studies of organisms as diverse as fungi (yeast), protozoa (Tetrahymena), higher plants (barley), echinoderms (sea urchin), insects (Musca), birds (chick) and mammals (mouse, rat). The technique is relatively insensitive to the use of harsh mechanical treatments for cell disruption or the presence of endogenous ribonuclease activity.     

Quantitative analysis of the protein composition of yeast ribosomes

The molecular weights of the individual yeast ribosomal proteins were determined. The ribosomal proteins from the 40-S subunit have molecular weights ranging from 11 800 to 31 000 (average molecular weight = 21 300). The molecular weights of the 60-S subunit proteins range from 10 000 to 48 400 (average molecular weight = 21 800). Stoichiometric measurements, performed by densitometric scanning on ribosomal proteins extracted from high-salt dissociated subunits revealed that isolated ribosomal subunits contain, besides some protein species occurring in submolar amounts, a number of protein species which are present in multiple copies: S13, S27, L22, L31, L33, L34 and L39. The mass fractions of the ribosomal proteins which were found to be present on isolated ribosomes in non-unimolar amounts, were re-examined by using an isotope dilution technique. Applying this method to proteins extracted from mildely isolated 80-S ribosomes, we found that some protein species such as S32, S34 and L43 still are present in submolar amounts. On the other hand, however, we conclude that some other ribosomal proteins, in particular the strongly acidic proteins L44 and L45 get partially lost during ribosome dissociation. Proteins L44/L45 appears to be present on 80-S ribosomes in three copies.     

Protein composition of Saccharomyces cerevisiae mitochondrial ribosomes

Protein composition of mitochondrial ribosomes of the yeast Saccharomyces cerevisiae was analysed by two-dimensional electrophoresis. The small (37S) mitoribosomal subunit contains 36 different polypeptides with molecular weights ranging from 10,000 to 60,000. The large (50S) subunit is composed of 41 proteins with molecular weights from 10,000 to 43,000. The molecular weights of mitoribosomal small and large subunits are 1.85 MDa and 2.35 MDa, respectively. Proteins represent 60-62% and 42-45% of the total mass of 37S and 50S subunits respectively. On the basis of the protein content and molecular weights of individual proteins we conclude that all mitoribosomal proteins are present in the mitoribosome in equimolar proportions.