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The limiting factors of a cell-free protein-synthesizing system from rat brain.

ABSTRACT: The limiting factors of a cell-free system from rat brain for incorporating amino acids into protein were studied. The initial more rapid incorporation by microsomes, as opposed to that by ribosomes, is suggested to be due to damage of the ribosomes by detergent. The defect is rectifiable by incubation of the ribosomes in cell sap, so that ribosomes eventually incorporate more amino acid than do microsomes. This may be because ribonuclease, which is associated with the microsomes but removed by detergent treatment, inactivates the microsomal system. The factor that causes incorporation by microsomes to cease abruptly within 1h is not the lack of any precursor or of adenosine triphosphate, of the inactivation of cell-sap factors or the accumulation of inhibitory substances, but is a deficiency of usable messenger ribonucleic acid. Chain initiation in the system is negligible. Ribosomes also become jammed at the end of messenger ribonucleic acid molecules, unable to terminate protein chains. This eventually leads to jammed polyribosomes, which can be partially relieved by very low concentrations of puromycin. A study of the release of polypeptides synthesized in response to the addition of synthetic messengers did not provide any conclusive information on chain-termination sequences, but did indicate some phenomena that were artifacts. It is concluded that ribonuclease action is sufficient to account for all the deficiencies of the cell-free system, but a lack of chain initiation may be a contributory factor.

SUBMITTER: Dunn AJ

PROVIDER: S-EPMC1185332 | biostudies-other | 1970 Jan

REPOSITORIES: biostudies-other

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Project description:1. Three procedures for isolating ribonucleoprotein particles from the cytoplasmic fraction of rat-uterus homogenates are described. By procedure 1, ribonucleoprotein particles were isolated in the presence of 5mm-Mg(2+) and 25mm-K(+), and the postmitochondrial supernatant fraction was made to 1.3% (w/v) in potassium deoxycholate. About 50% of the RNA and protein of the microsomal fraction was recovered in the monomeric ribosomes isolated. By procedure 2, ribonucleoprotein particles were isolated in the presence of 10mm-Mg(2+) and 0.1m-K(+), and in the absence of detergent. The ribosomes obtained were primarily polymeric, but recovery of microsomal RNA and protein was only 32%. By procedure 3, ribonucleoprotein particles were isolated according to procedure 1 but without the use of detergent. A mixture of polymeric and monomeric ribosomes was obtained, and the recovery of microsomal RNA and protein was about 60%. 2. Uterine polymeric and monomeric ribosomes, isolated by procedure 3 and designated ;polyribosomal preparation', were examined for protein-synthesizing capabilities. The principal properties of the cell-free protein-synthesizing system containing the polyribosomal preparation are described. The efficiency of amino acid incorporation in the complete system incubated for 30min. and containing the polyribosomal preparation was found to be either 2.5 molecules of [(14)C]leucine or 2.2 molecules of [(14)C]-valine incorporated/ribosome. Assay of the preparation in the complete cell-free system containing 10mm-sodium fluoride indicated that 40% of the incorporation activity is a result of initiation of new polypeptide chains and 60% is due to completion of previously existing chains. Monomeric ribosomes obtained by various treatments of the polyribosomal preparation with sodium fluoride, ribonuclease and potassium deoxycholate had decreased incorporation activity in the cell-free system. However, monomeric ribosomes obtained by treatment with sodium fluoride only had an incorporation activity 50% greater than that of monomers obtained by treatment with ribonuclease only. 3. The results indicate that uterine polymeric and monomeric ribosomes are sites of amino acid incorporation in vivo and in vitro. It is concluded that most polymeric and monomeric ribosomes occurring in the cytoplasmic fraction of the uterus are free and unattached to membranes, and that the polyribosomes are relatively unstable.

Project description:1. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the presence and absence of polyuridylic acid. 2. The activity of a light-microsome fraction from adult liver was greater than that of a heavy-microsome fraction, and the light-microsome fraction was also more markedly stimulated by the presence of polyuridylic acid. 3. The light-microsome fraction, when analysed by density-gradient centrifugation, contained a higher ratio of free ribosomes to bound ribosomes, whereas the reverse was true for the heavy-microsome fraction. Similar results were obtained for liver from adult and 5-day-old rats. 4. When the light-microsome fraction was incubated under conditions in which amino acid was incorporated into protein there was only a small increase in the ratio of free to bound ribosomes. When such a fraction was incubated with [(14)C]leucine and was then subjected to density-gradient centrifugation the fraction with the highest specific activity based on RNA had a density between that of the bound and free ribosomes. Treatment of the incubated fraction with ribonuclease shifted the radioactivity towards the free ribosome peak. These properties are consistent with the presence of active free polysomes. Such a component appeared also to be present when the heavy-microsome fraction was incubated under similar conditions. 5. The effect of the presence of polyuridylic acid on the incorporation of [(14)C]phenylalanine by the light-microsome fractions from liver of adult and 5-day-old rats was greatest in the region of the free ribosomes, but it is probable that some small polysomes containing polyuridylic acid are formed. 6. Polyuridylic acid also stimulated the bound ribosomes to a small extent when the heavy-microsome fraction from the liver of young rats was incubated with [(14)C]phenylalanine. 7. The results are discussed in terms of the various morphological constituents in liver now known to play a role in the synthesis of protein for export and for the internal activity of the cell.

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The limiting factors of a cell-free protein-synthesizing system from rat brain.

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