Studies on protein biosynthesis

McLean, Elizabeth J. T (1962) Studies on protein biosynthesis. PhD thesis, University of Glasgow.

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1. Protein Depletion causes pronounced loss of RNA from the liver cell, a loss which is associated predominantly with the endoplasmic reticulum. We have shown that, during this period of acute RNA breakdown, the RNA content of the cell sap is increased. Evidence has been presented suggesting that the RNA breakdown products from the endoplasmic reticulum accumulate in the cell sap. 2. The capacity of the cell sap RNA to bind amino acids is also altered by diet but varies inversely with the RNA present. When, in response to protein depletion, the RNA content increases, the incorporation of leucine is correspondingly diminished. However, although the amount of cell sap RNA changes with protein intake, the total capacity to accept amino acids is unchanged. Thus the additional RNA found in the cell sap appears to be insert as an amino acid acceptor and merely dilutes leucine incorporation by the sRNA present. 3. The effects produced on the cell sap RNA by the dietary conditions are due to changes in the post-microsomal pellet, a particulate fraction which is sedimented by centrifuging the cell sap for 3 hrs. at 105,000 g. As in the cell sap, the capacity of the post-microsomal pellet to accept amino acids and the amount of RNA present vary inversely with diet. 4. RNA purified from the post-microsomal pellet was inert as an amino acid acceptor, even in the presence of rat liver pH 5 enzyme. sRNA was therefore presumed to be absent from the post-microsomal pellet; this was confirmed by column chromatography. 5. In the presence of an energy source, the post-microsomal pellet is capable of incorporating leucine into a form which is stable to hot-acid extraction and not therefore associated with RNA. Only a small proportion of the total leucine incorporated was solubilised by this treatment. 6. The apparent existence of an sRNA free mechanism for amino acid incorporation prompted an investigation of the amino acid uptake observed in the post-microsomal pellet. 7. The leucine uptake into the hot-acid-stable fraction of the post-microsomal pellet is a. dependant on ATP as an energy source. b. inhibited by GTP, CTP and UTP. c. stimulated by amino acid mixtures in the presence of all four nucleoside triphosphates. d. inhibited by glutathione. e. unaffected by chloramphenicol. f. unaffected by ribonuclease (possibly even augmented) in sufficient concentration to reduce the RNA present to 8% of its original value. g. inhibited by addition of pH 5 enzyme from the cell sap. 8. The leucine uptake cannot be equated to complete protein synthesis since the incorporated amino acid is located in a terminal position from which it may easily be displaced in the presence of excess leucine. Other amino acids are incorporated to a reduced extent or not at all. 9. The total activating enzyme activity in the post-microsomal pellet as judged by amino acid dependent ATP-32P pyrophosphate exchange is almost equal to the level of these enzymes found in the cell sap. A large proportion of the enzyme present is specific for leucine. However, if the activated acyl-adenylate complex for leucine is formed, in the post-microsomal pellet, it appears to be unable to donate the leucine to sRNA, the natural acceptor. 10. The behaviour of the post-microsomal pellet is not in accordance with the mechanism described by Hoagland for protein synthesis from free amino acids and indeed cannot be reconciled with any step in the complicated pathway involved in amino acid activation and formation into peptide chains. The relationship of this system to various protein synthetic mechanisms proposed in the literature is discussed.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: H N Munro
Keywords: Biochemistry
Date of Award: 1962
Depositing User: Enlighten Team
Unique ID: glathesis:1962-73316
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56

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