The effect of ribonuclease on rat-liver ribosomes

1. Rat-liver ribosomes lose about 50% of their amino acid-incorporating activity when preincubated with ribonuclease. 2. This preincubation results also in loss of about 50% of the original protein content and 75% of the RNA. 3. Ribosomes sedimented by ultracentrifugation, after preincubation with ribonuclease, show negligible contamination by crystalline enzyme. 4. Washing of ribosomes treated with ribonuclease releases further protein, restoring the original RNA/protein ratio. 5. The washed particle is again capable of promoting amino acid incorporation. 6. Examination of ribosomes treated with ribonuclease in the analytical ultracentrifuge reveals destruction of ribosomes, disappearance of dimers and a decrease in the sedimentation coefficient of monomers. 7. Washed ribosomes consist of even smaller particles with a sedimentation coefficient 60s.     

The isolation and properties of cardiac ribosomes and polysomes

1. A method is described by which good yields of ribosomes and polysomes free of contamination by submitochondrial fragments can be prepared from rat cardiac muscle. These preparations are capable of incorporation of amino acids into protein in vitro. 2. The ribosome preparation consists of 32% of monomeric ribosomes and 68% of ribosomal aggregates or polysomes. The polysome preparation has a decreased monomeric content. Dimers, trimers, tetramers, pentamers and larger components can be differentiated. 3. The polysome aggregate structure is degraded to monomeric ribosomes on incubation with small amounts of ribonuclease or by preparation in the absence of Mg²⁺ ions. The degradation in the absence of Mg²⁺ ions was not reversible and drastically decreased the incorporation of amino acids in vitro. 4. The cardiac ribosomes contained two major RNA species sedimenting at 19s and 28s in a 1:2·4 ratio. 5. The RNA/protein ratio of cardiac ribosomes and polysomes was consistently lower than that of similar preparations from liver. The concentrations of Na⁺ and K⁺ ions present during preparation had a great effect on the RNA/protein ratio. 6. Optimum conditions for the incorporation of amino acids into protein in vitro are reported. Cardiac ribosomes have a lower rate of incorporation of amino acids in vitro than liver ribosomes. 7. Heart cell sap is less active than liver cell sap: evidence is presented that a factor, present in liver cell sap and concerned with stimulating the synthesis of the peptide chain, is lacking in heart cell sap. 8. Pulse-labelling of perfused hearts followed by examination of the subcellular structures showed that the ribosomal fraction was the most active in the incorporation of amino acids in vitro.     

Action of ribonuclease upon the polysomes of cultured mouse cells

Summary Pancreatic ribonuclease (RNase) and³H-uridine were used to study certain compositional and ontogenetic features of the polysomes of strain L mouse cells. Growing cells were exposed to the radioactive nucleoside,³H-uridine, for brief defined periods, and the sensitivity of the polysomes to digestion by RNase was determined. The RNase-resistant RNA of polysomes is shown to be primarily ribosomal, and the RNase-sensitive material formed during brief pulse labeling studies is largely messenger RNA. Actinomycin D inhibition of RNA synthesis was used to confirm this identification. The technique described here was used to investigate the effects of hydrocortisone on polysome formation. The hormone (10⁻⁶ m) lessens the extent of the nucleoside incorporation into polysomal and total RNA and delays the appearance of newly synthesized 18 S and 28 S rRNA into cytoplasmic polysomes. This work was partially supported by grants from the United States Public Health Service (GM 10866), from the National Science Foundation (GB 13924), and from The University of Kansas General Research Fund.     

The inhibition of ribosomal ribonuclease by bacterial ribosomes

1. A comparison has been made between the ribonuclease activities of untreated ribosomes from Escherichia coli B and Pseudomonas fluorescens and the activities of ribosomes on to which ribosomal ribonuclease from E. coli B has been adsorbed. 2. The normal ribosomes from both species were stable in 5-10mm-Mg(2+) (I0.16) at pH6. The RNA in ribosomes from Ps. fluorescens was attacked by the adsorbed ribonuclease under these conditions, whereas the ribosomes from E. coli B were able to adsorb and inhibit this enzyme. 3. Inhibition was also observed with ribosomes from Aerobacter aerogenes, Proteus vulgaris and two other strains of E. coli. It was not observed in ribosomes from three species of Pseudomonas. 4. The inhibition depended on the integrity of the ribosomes and was not observed under conditions of low Mg(2+) concentration that cause irreversible degradation into more slowly sedimenting particles.     

Hydrophobic binding of ribosomes and polysomes to agarose gels

Rat liver ribosomes and polyribosomes could be immobilized in agarose gels at 4°C and pH 7.6, using KCl or NaCl molarities of 0.25 or higher. The binding could be effected in the presence of excess protein and/or detergents. Polysomes attached to endoplasmic membrane fragments did not bind to agarose even at 0.5m KCl; tRNAs were also not bound. The larger (60 S) subunit of liver ribosomes was also completely immobilized at 0.3m KCl, while the immobilization of the smaller (40 S) subunit was poor even at 1m KCl. The ribosomal subunits could be essentially quantitatively desorbed at 4°C by a low ionic strength elution, while the recovery of gel-bound polysomes was of the order of 80 to 85% under these conditions. The polysomes that recovered from agarose at low ionic strength were active inin vitro incorporation of amino acids into polypeptides.     

The effect of pancreatic ribonuclease on rabbit reticulocyte ribosomes and its interpretation in terms of ribosome structure

1. Parts of the 16s and 30s RNA species of reticulocytes are readily hydrolysed by pancreatic ribonuclease. The biological activity of the ribosomes is diminished after treatment with low concentrations of the enzyme (e.g. 1ng. of ribonuclease/2.5mg. of polyribosome fraction/ml.). A high proportion of the chain scissions are ;hidden' owing to the secondary structure of the RNA moiety. 2. As the concentration of ribonuclease is increased RNA is lost from the ribosome. About 20-30% of the RNA may be removed from the ribosome without altering appreciably its sedimentation coefficient or its appearance in the electron microscope. 3. The amount of RNA removed from the ribosome is not increased by raising the concentration of enzyme from about 1mug. to 2.5mg. of ribonuclease/2.5mg. of polyribosome fraction/ml., or by increasing the temperature from 0 degrees to 30 degrees , or by first converting the RNA moiety into a single-stranded form before exposure to ribonuclease. 4. Untreated polyribosomes aggregate at about 75 degrees , whereas ribosomes treated with ribonuclease aggregate at about 45 degrees . The aggregates that are found on heating ribosomes after enzymic hydrolysis contain about 40-50% of the complement of RNA of intact ribosomes. 5. From the size of the fragments of RNA isolated from RNA-depleted ribosomes it is inferred that there is one site/60-100 nucleotides that is sensitive to ribonuclease. 6. The RNA moiety of RNA-depleted ribosomes has some double-helical character as shown by the optical properties and X-ray-diffraction pattern of ribonuclease-treated ribosomes and by the ;melting' properties of the isolated RNA. 7. Subparticles prepared by titration with an excess of EDTA are readily hydrolysed by ribonuclease to fragments of S(20,w) less than 4s, in contrast with the intact particle.     

Physiology of rat-liver polysomes: The stability of messenger ribonucleic acid and ribosomes

Starvation of rats for several days led to marked decrease in cytoplasmic polysomes and accumulation of breakdown products having S values less than 200s. Re-feeding of the starved animals induced a rapid reassembly of polysomes. These newly formed polysomes, in the presence of actinomycin D, decayed in a biphasic fashion: about two-thirds decayed with an apparent half-life of 3-3(1/2)hr. but the other one-third were much more stable. Evidence that polysome decay is an accurate reflexion of messenger RNA stability is presented, and it is concluded that in the presence of large doses of actinomycin D, rat-liver cytoplasm contains messenger RNA classes of widely varying stability, the more stable class having a half-life of at least 80hr. The half-life of liver ribosomes was also determined and was found to be 110-127hr.