The action of ricin A chain on eukaryotic ribosomes was investigated from a number of different angles. It was shown that ricin A chain modified the rRNA from the 60S subunit of a number of eukaryotic ribosomes, including plant ribosomes, and that the site of action was at the same position in a highly conserved sequence which had previously been identified as the site of action in rat liver 28S rRNA. Investigations into the partial reactions of protein synthesis inhibited in ricin A chain-treated ribosomes showed that both initiation and elongation were inhibited, contradicting the assumption based on previous work that ricin A chain inhibited just the elongation cycle. In a rabbit reticulocyte lysate it was found that whilst elongation was severely inhibited by ricin A chain, the rate of initiation was also reduced approximately six-fold relative to that seen with an inhibitor of just elongation. Furthermore, experiments carried out to investigate the inhibition of elongation showed that this was most likely due to the inhibition of the elongation factor 2 catalysed step. Using an assay which allowed the N-glycosidase activity of ricin A chain to be measured directly it was possible to show that ribosomes from a number of different sources varied markedly in their sensitivity to ricin A chain. Wheat germ ribosomes were shown to be in the order of 1000 times less sensitive to modification than those from either yeast or rabbit reticulocytes. However, this difference does not seem to be a reflection of the differential affinity of ricin A chain for the various substrates but rather a consequence of the ability of the toxin to modify the rRNA once it has bound. This is because kinetic experiments showed that the Km for the reaction on wheat germ ribosomes was similar to that which had been published for the action of ricin A chain on rat liver or rabbit ribosomes. The Kcat, however, was approximately 3 orders of magnitude smaller. A similar picture was seen with the type 1 RIP dianthin 32. It was shown directly that elongation factor 2 bound irreversibly to the ribosome could protect the ribosome from the action of ricin A chain and that in a wheat germ lysate this ability to compete out ricin A chain seemed to be a property of just this supernatant protein. This protection is reflected in the finding that the elongation factor 2 content of purified ribosomes determines their sensitivity to depurination by ricin A chain and that the removal of this protein with high salt sensitises the ribosomes to modification.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:305195 |
| Date | January 1990 |
| Creators | Osborn, Rupert W. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/108875/ |
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